Compositions for saccharification of cellulosic material

ABSTRACT

The present invention relates to enzyme compositions for high temperature saccharification of cellulosic material and to uses thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/115,930, filed Aug. 29, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/422,245 filed Feb. 1, 2017, now U.S. Pat. No.10,072,280, which is a divisional of U.S. patent application Ser. No.14/885,555 filed Oct. 16, 2015, now U.S. Pat. No. 9,587,262, which is adivisional of U.S. patent application Ser. No. 14/052,360 filed Oct. 11,2013, now U.S. Pat. No. 9,175,277, which is a divisional of U.S. patentapplication Ser. No. 12/940,952 filed Nov. 5, 2010, now U.S. Pat. No.8,580,536, which claims the benefit of U.S. Provisional Application Ser.No. 61/259,014 filed Nov. 6, 2009, which are incorporated herein byreference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support underCooperative Agreement DE-FC36-08G018080 awarded by the Department ofEnergy. The government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO DEPOSITS OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biologicalmaterial, which deposits are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to enzyme compositions for hightemperature saccharification of cellulosic material and to uses thereof.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4bonds. Many microorganisms produce enzymes that hydrolyze beta-linkedglucans. These enzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

There is a need in the art for new enzyme compositions to increaseefficiency and to provide cost-effective enzyme solutions for hightemperature saccharification of cellulosic material.

The present invention provides compositions for high temperaturesaccharification of cellulosic material and to uses thereof.

SUMMARY OF THE INVENTION

The present invention relates to enzyme compositions, comprising two ormore (several) components selected from the group consisting of:

(I) a polypeptide having cellobiohydrolase I activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 3, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 3;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 6; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 5;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 8; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 7, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 7;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 158; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 157, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 157, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 157;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 160; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 159, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 159, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 159;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 162; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 161, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 161, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 161;

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 164; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 163, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 163, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 163; and

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 166; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 165, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 165, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 165;

(II) a polypeptide having cellobiohydrolase II activity selected fromthe group consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 10; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 9, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 9, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 9;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 12; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 11, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 11, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 11;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 14; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 13, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 13, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 13;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 16; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 15, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 15, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 15;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 17, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 17, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 17;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 168; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 167, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 167, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 167;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 170; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 169, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 169, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 169; and

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 172; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 172, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 172, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 172;

(III) a polypeptide having endoglucanase I activity selected from thegroup consisting of: (a) a polypeptide comprising an amino acid sequencehaving preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 20; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 19, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 19, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 19;

(IV) a polypeptide having endoglucanase II activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 22; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 21, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 21, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 21;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 24; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 23, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 23, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 23;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 26; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 25, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 25, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 25;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 174; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 173, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 173, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 173; and

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 176; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 175, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 175, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 175; and

(V) a polypeptide having beta-glucosidase activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 28; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 27, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 27, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 27;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 30; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 29, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 29, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 29;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 32; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 31, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 31, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 31;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 178; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 177, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 177, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 177;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 180; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 179, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 179, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 179;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 182; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 181, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 181, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 181;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 184; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 183, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 183, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 183;

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 186; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 185, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 185, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 185;

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 188; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 187, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 187, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 187; and

(J) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 190; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 189, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 189, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 189.

The present invention also relates to host cells encoding such an enzymecomposition and methods of producing such an enzyme composition.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with such an enzyme composition.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with such an enzyme composition;

(b) fermenting the saccharified cellulosic material with one or more(several) fermenting microorganisms to produce the fermentation product;and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with such an enzyme composition.

BRIEF DESCRIPTION OF THE FIGS

FIG. 1 shows a comparison of two enzyme compositions with a Trichodermareesei-based composition in hydrolysis of milled washed PCS at 50° C.,55° C., and 60° C.

FIG. 2 shows the effect of Thermoascus aurantiacus GH61A or Thielaviaterrestris GH61E GH61 polypeptides having cellulolytic enhancingactivity on PCS-hydrolysing activity of a high-temperature enzymecomposition at 50° C., 55° C., and 60° C.

FIG. 3 shows the boosting performance of a binary composition comprisingequal amounts of Thermoascus aurantiacus GH61A and Thielavia terrestrisGH61E GH61 polypeptides having cellulolytic enhancing activity incomparison with the boosting performance of the individual GH61polypeptides in hydrolysis of milled washed PCS at 50° C., 55° C., and60° C.

FIG. 4 shows the effect of compositions containing different ratios ofThermoascus aurantiacus GH61A and Thielavia terrestris GH61Epolypeptides on PCS-hydrolysing activity of a high-temperature enzymecomposition at 60° C.

FIG. 5 shows the effect of different levels of individual Thermoascusaurantiacus GH61A and Thielavia terrestris GH61E GH61 polypeptideshaving cellulolytic enhancing activity and their binary 1:1 compositionon PCS-hydrolyzing activity of a high-temperature enzyme composition at60° C.

FIG. 6 shows the effect of a Thermobifida fusca GH11 xylanase onhydrolysis of milled washed PCS by a high-temperature enzyme compositionat 50-65° C.

FIG. 7 shows the effect of replacing Chaetomium thermophilum Cel7Acellobiohydrolase I in a high-temperature enzyme composition withvarious thermostable cellobiohydrolase I proteins on hydrolysis ofmilled washed PCS at 50-65° C.

FIG. 8 shows a comparison of Aspergillus fumigatus Cel7A- and Chaetomiumthermophilum Cel7A-based high-temperature enzyme compositions withTrichoderma reesei-based cellulase XCL-533 at 50° C. and 60° C. inhydrolysis of milled washed PCS.

FIG. 9 shows the hydrolysis time-course for Aspergillus fumigatusCel7A-based high-temperature enzyme composition in comparison withTrichoderma reesei-based cellulase XCL-533 at 50° C. and 60° C. (2 mgprotein/g cellulose).

FIG. 10 shows an evaluation of Aspergillus aculeatus GH10 xylanase II,Aspergillus fumigatus GH10 xyn3 xylanase, Trichophaea saccata GH10xylanase, and Thermobifida fusca GH11 xylanase at 10% addition (0.35 mgprotein/g cellulose) to a high-temperature enzyme composition (3.5 mgprotein/g cellulose) in hydrolysis of milled washed PCS at 50° C., 55°C., and 60° C.

FIG. 11 shows an evaluation of Aspergillus fumigatus GH10 xyn3 xylanase,Trichophaea saccata GH10 xylanase, and Thermobifida fusca GH11 xylanasefor synergy with a high-temperature enzyme composition in hydrolysis ofmilled washed PCS at 50° C., 55° C., and 60° C. Each xylanase was addedat different levels (1.25%, 2.5%, 5%, 10%, and 20%) to a constantloading of the high-temperature enzyme composition (3 mg protein per gcellulose).

FIG. 12 shows a comparison of an improved high-temperature enzymecomposition containing Aspergillus fumigatus GH10 xyn3 xylanase at 60°C. with Trichoderma reesei-based cellulase XCL-533 at 50° C. inhydrolysis of milled washed PCS.

FIGS. 13A and 13B show a comparison of improved high-temperature enzymecompositions containing Aspergillus fumigatus GH10 xyn3 xylanase orTrichophaea saccata GH10 xylanase (60° C.) with Trichoderma reesei-basedcellulase XCL-533 (50° C.) in hydrolysis of washed (A) and unwashed (B)PCS.

FIGS. 14A and 14B show the effect of replacement of protein in ahigh-temperature enzyme composition (3 mg protein per g cellulose) withGH3 beta-xylosidases from Trichoderma reesei and Talaromyces emersoniiat 60° C.

FIG. 15 shows a comparison of Trichoderma reesei Cel7A CBHI, Chaetomiumthermophilum Cel7A CBHI, Aspergillus fumigatus Cel7A CBHI, andThermoascus aurantiacus Cel7A CBHI replacing a CBHI component in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 16 shows a comparison of Myceliophthora thermophila Cel6A CBHII,Thielavia terrestris Cel6A CBHII, Aspergillus fumigatus Cel6A CBHII, andTrichophaea saccata Cel6A CBHII replacing a CBHII component in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 17 shows a comparison of Trichoderma reesei Cel7B EGI andAspergillus terreus Cel7 EGI replacing an endoglucanase component in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 18 shows a comparison of Trichoderma reesei Cel5A EGII,Myceliophthora thermophila Cel5A EGII, and Thermoascus aurantiacus Cel5AEGII replacing an endoglucanase component in a high-temperature enzymecomposition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 19 shows a comparison of Aspergillus fumigatus Cel3Abeta-glucosidase, Penicillium brasilianum Cel3A beta-glucosidase, andAspergillus niger Cel3 beta-glucosidase in a high-temperature enzymecomposition at 50-60° C. using milled unwashed PCS.

FIG. 20 shows a comparison of Aspergillus fumigatus Cel3Abeta-glucosidase, Penicillium brasilianum Cel3A beta-glucosidase, andAspergillus niger Cel3 beta-glucosidase in a high-temperature enzymecomposition at 50-65° C. using milled unwashed PCS.

FIG. 21 shows a comparison of Aspergillus aculeatus GH10 xyn IIxylanase, Aspergillus fumigatus GH10 xyn3, Trichophaea saccata GH10xylanase, Thermobifida fusca GH11 xylanase, Penicillium pinophilum GH10xylanase, and Thielavia terrestris GH10E xylanase replacing a xylanasecomponent in a high-temperature enzyme composition in hydrolysis ofmilled unwashed PCS at 50-65° C.

FIG. 22 shows a comparison of the cellulase-enhancing activity ofThermoascus aurantiacus GH61A, Thielavia terrestris GH61E, Penicilliumpinophilum GH61, and Aspergillus fumigatus GH61B polypeptides replacinga GH61 component in a high-temperature enzyme composition in hydrolysisof milled unwashed PCS at 50-65° C.

FIG. 23 shows a comparison of the cellulase-enhancing activity ofThermoascus aurantiacus GH61A, Thielavia terrestris GH61N, andPenicillium sp GH61A polypeptides replacing a GH61 component in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 24 shows the effect of Trichoderma reesei-based XCL-602 cellulasereplacement by Aspergillus fumigatus Cel7A cellobiohydrolase I and/orMyceliophthora thermophila Cel6A cellobiohydrolase II onsaccharification of milled unwashed PCS at 50-60° C.

FIGS. 25A and 25B show the hydrolysis of milled unwashed PCS byTrichoderma reesei-based XCL-602 cellulase compositions containingAspergillus fumigatus Cel7A cellobiohydrolase I and Myceliophthorathermophila Cel6A cellobiohydrolase II (3 mg total protein per gcellulose) and additionally supplemented by 5% Aspergillus fumigatusGH10 xyn 3 and/or 5% Thielavia terrestris GH61E at 50-60° C.

FIG. 26 shows the hydrolysis of milled unwashed PCS by Trichodermareesei-based XCL-602 compositions containing different replacementlevels of Trichoderma reesei-based XCL-592 cellulase at 50-60° C.

FIGS. 27A and 27B show a comparison of Thermoascus aurantiacus GH61A andThielavia terrestris GH61E polypeptides replacing 5% of protein inTrichoderma reesei-based XCL-602 cellulase or XCL-602-based enzymecomposition in hydrolysis of milled unwashed PCS at 50-60° C.

FIGS. 28A and 28B show the hydrolysis of milled unwashed PCS bynon-replaced Trichoderma reesei-based XCL-602 cellulase and variousXCL-602-based enzyme compositions (3 mg protein per g cellulose) incomparison with Trichoderma reesei-based XCL-533 cellulase (4.5 mgprotein per g cellulose) at 50-60° C.

FIG. 29 shows a comparison of Aspergillus fumigatus Cel7A CBHI andPenicillium emersonii Cel7 CBHI in a high-temperature enzyme compositionin hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 30 shows an evaluation of Aspergillus fumigatus Cel7A CBHI andPenicillium pinophilum Cel7A CBHI in a high-temperature enzymecomposition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 31 shows an evaluation of Aspergillus fumigatus Cel7A CBHI andAspergillus terreus Cel7A CBHI in a high-temperature enzyme compositionin hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 32 shows an evaluation of Aspergillus fumigatus Cel7A CBHI,Neosartorya fischeri Cel7A CBHI, and Aspergillus nidulans Cel7A CBHI ina high-temperature enzyme composition in hydrolysis of milled unwashedPCS at 50-60° C.

FIG. 33 shows an evaluation of Aspergillus fumigatus Cel6A CBHII andFinnellia nivea Cel6A CBHII in a high-temperature enzyme composition inhydrolysis of milled unwashed PCS at 50-65° C.

FIG. 34 shows an evaluation of Aspergillus fumigatus Cel6A CBHII,Penicillium emersonii Cel6A CBHII, and Penicillium pinophilum Cel6ACBHII proteins replacing a CBHII component in a high-temperature enzymecomposition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 35 shows an evaluation of Aspergillus fumigatus Cel5A EGII,Neosartorya fischeri Cel5A EGII, and Myceliophthora thermophila Cel5AEGII proteins replacing a EG component in a high-temperature enzymecomposition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 36 shows an evaluation of Aspergillus fumigatus Cel3Abeta-glucosidase and Aspergillus aculeatus beta-glucosidase in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 37 shows an evaluation of Aspergillus fumigatus Cel3Abeta-glucosidase, Aspergillus kawashii Cel3A beta-glucosidase,Aspergillus clavatus Cel3 beta-glucosidase, and Talaromyces emersoniiCel3A beta-glucosidase in a high-temperature enzyme composition inhydrolysis of milled unwashed PCS at 50-60° C.

FIG. 38 shows an evaluation of Aspergillus fumigatus Cel3Abeta-glucosidase, Penicillium oxalicum Cel3A beta-glucosidase (Example77) and Penicillium oxalicum Cel3A beta-glucosidase (Example 78) in ahigh-temperature enzyme composition in hydrolysis of milled unwashed PCSat 50-65° C.

FIG. 39 shows an evaluation of three GH61 polypeptides havingcellulolytic enhancing activity in a high-temperature enzyme compositionin hydrolysis of milled washed PCS at 50-65° C.

FIG. 40 shows an evaluation of three xylanases in a high-temperatureenzyme composition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 41 shows an evaluation of three xylanases in a high-temperatureenzyme composition in hydrolysis of milled unwashed PCS at 50-65° C.

FIG. 42 shows the hydrolysis of milled unwashed PCS by non-replacedTrichoderma reesei-based XCL-602 cellulase and various XCL-602-basedenzyme compositions containing different cellobiohydrolases andxylanases (3 mg protein per g cellulose) at 50-60° C.

DEFINITIONS

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman N21filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman N21filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-β-D-lactoside.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis ofterminal non-reducing beta-D-glucose residues with the release ofbeta-D-glucose. For purposes of the present invention, beta-glucosidaseactivity is determined according to the basic procedure described byVenturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomiumthermophilum var. coprophilum: production, purification and somebiochemical properties, J. Basic Microbiol. 42: 55-66. One unit ofbeta-glucosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at 50° C. compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5L (Novozymes A/S, Bagsvrd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (several) enzymes thathydrolyze a hemicellulosic material. See, for example, Shallom, D. andShoham, Y. Microbial hemicellulases. Current Opinion In Microbiology,2003, 6(3): 219-228). Hemicellulases are key components in thedegradation of plant biomass. Examples of hemicellulases include, butare not limited to, an acetylmannan esterase, an acetyxylan esterase, anarabinanase, an arabinofuranosidase, a coumaric acid esterase, aferuloyl esterase, a galactosidase, a glucuronidase, a glucuronoylesterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. Thesubstrates of these enzymes, the hemicelluloses, are a heterogeneousgroup of branched and linear polysaccharides that are bound via hydrogenbonds to the cellulose microfibrils in the plant cell wall, crosslinkingthem into a robust network. Hemicelluloses are also covalently attachedto lignin, forming together with cellulose a highly complex structure.The variable structure and organization of hemicelluloses require theconcerted action of many enzymes for its complete degradation. Thecatalytic modules of hemicellulases are either glycoside hydrolases(GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs),which hydrolyze ester linkages of acetate or ferulic acid side groups.These catalytic modules, based on homology of their primary sequence,can be assigned into GH and CE families marked by numbers. Somefamilies, with overall similar fold, can be further grouped into clans,marked alphabetically (e.g., GH-A). A most informative and updatedclassification of these and other carbohydrate active enzymes isavailable on the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure &Appl. Chem. 59: 1739-1752.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TritonX-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6 buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%Triton X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom the non-reducing termini. For purposes of the present invention,one unit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyses the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20. One unit of acetylxylan esterase is definedas the amount of enzyme capable of releasing 1 μmole of p-nitrophenolateanion per minute at pH 5, 25° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Family 3, 5, 6, 7, 10, 11, or 61, or GH3, GH5, GH6, GH7, GH10, GH11, orGH61, or Cel3, Cel5, Cel6 or Cel7: The terms “Family 3”, “Family 5”,“Family 6”, “Family 7”, “Family 10”, “Family 11”, “Family 61”, “GH3”,“GH5”, “GH6”, “GH7”, “GH10”, “GH11”, “GH61”, “Cel3”, “Cel5”, “Cel6”, or“Cel7” are defined herein as a polypeptide falling into the glycosidehydrolase Families 3, 5, 6, 7, 10, 11, and 61 according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino-acidsequence similarities, Biochem. J. 280: 309-316, and Henrissat andBairoch, 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Cellulosic material: The cellulosic material can be any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is lignocellulose.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is corn fiber. In another aspect, thecellulosic material is corn cob. In another aspect, the cellulosicmaterial is orange peel. In another aspect, the cellulosic material isrice straw. In another aspect, the cellulosic material is wheat straw.In another aspect, the cellulosic material is switch grass. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is bagasse. In another aspect, the cellulosicmaterial is softwood. In another aspect, the cellulosic material ishardwood.

In another aspect, the cellulosic material is microcrystallinecellulose. In another aspect, the cellulosic material is bacterialcellulose. In another aspect, the cellulosic material is algalcellulose. In another aspect, the cellulosic material is cotton linter.In another aspect, the cellulosic material is amorphous phosphoric-acidtreated cellulose. In another aspect, the cellulosic material is filterpaper.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid.

Isolated or purified: The term “isolated” or “purified” means apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, apolypeptide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, or at least 95%pure, as determined by agarose electrophoresis.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. The mature polypeptide can bepredicted using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6).

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having biological activity. The mature polypeptidecoding sequence can be predicted using the SignalP program (Nielsen etal., 1997, supra).

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Polypeptide fragment: The term “fragment” means a polypeptide having oneor more (several) amino acids deleted from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has biologicalactivity.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving biological activity.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptide.Each control sequence may be native or foreign to the polynucleotideencoding the polypeptide or native or foreign to each other. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence. Expression: Theterm “expression” includes any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

DETAILED DESCRIPTION OF THE INVENTION Enzyme Compositions

The present invention relates to enzyme compositions, comprising two ormore (several) components selected from the group consisting of:

(I) a polypeptide having cellobiohydrolase I activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 1;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 3, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 3;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 6; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 5;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 8; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 7, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 7;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 158; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 157, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 157, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 157;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 160; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 159, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 159, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 159;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 162; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 161, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 161, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 161;

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 164; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 163, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 163, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 163; and

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 166; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 165, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 165, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 165;

(II) a polypeptide having cellobiohydrolase II activity selected fromthe group consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 10; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 9, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 9, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 9;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 12; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 11, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 11, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 11;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 14; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 13, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 13, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 13;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 16; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 15, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 15, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 15;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 17, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 17, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 17;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 168; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 167, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 167, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 167;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 170; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 169, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 169, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 169; and

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 172; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 172, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 172, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 172;

(III) a polypeptide having endoglucanase I activity selected from thegroup consisting of: (a) a polypeptide comprising an amino acid sequencehaving preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 20; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 19, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 19, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 19;

(IV) a polypeptide having endoglucanase II activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 22; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 21, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 21, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 21;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 24; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 23, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 23, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 23;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 26; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 25, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 25, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 25;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 174; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 173, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 173, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 173; and

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 176; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 175, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 175, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 175; and

(V) a polypeptide having beta-glucosidase activity selected from thegroup consisting of:

(A) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 28; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 27, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 27, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 27;

(B) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 30; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 29, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 29, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 29;

(C) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 32; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 31, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 31, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 31;

(D) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 178; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 177, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 177, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 177;

(E) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 180; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 179, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 179, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 179;

(F) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 182; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 181, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 181, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 181;

(G) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 184; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 183, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 183, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 183;

(H) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 186; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 185, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 185, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 185;

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 188; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 187, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 187, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 187; and

(J) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 190; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 189, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 189, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 189.

In a preferred aspect, the polypeptide having cellobiohydrolase Iactivity is a Chaetomium thermophilum Cel7A cellobiohydrolase I of themature polypeptide of SEQ ID NO: 2. In another aspect, the Chaetomiumthermophilum Cel7A cellobiohydrolase I is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 1.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Myceliophthora thermophila Cel7A cellobiohydrolase I of the maturepolypeptide of SEQ ID NO: 4. In another aspect, the Myceliophthorathermophila Cel7A cellobiohydrolase I is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 3.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Aspergillus fumigatus Cel7A cellobiohydrolase I of the maturepolypeptide of SEQ ID NO: 6. In another aspect, the Aspergillusfumigatus Cel7A cellobiohydrolase I is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 5.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Thermoascus aurantiacus Cel7A cellobiohydrolase I of the maturepolypeptide of SEQ ID NO: 8. In another aspect, the Thermoascusaurantiacus Cel7A cellobiohydrolase I is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 7.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Penicillium emersonii Cel7 cellobiohydrolase I of the maturepolypeptide of SEQ ID NO: 158. In another aspect, the Penicilliumemersonii Cel7 cellobiohydrolase 1 is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 157.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis a Penicillium pinophilum Cel7 cellobiohydrolase 1 of the maturepolypeptide of SEQ ID NO: 160. In another aspect, the Penicilliumpinophilum Cel7A cellobiohydrolase 1 is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 159.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis an Aspergillus terreus Cel7 cellobiohydrolase 1 of the maturepolypeptide of SEQ ID NO: 162. In another aspect, the Aspergillusterreus Cel7 cellobiohydrolase 1 is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 161.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis a Neosartorya fischeri Cel7 cellobiohydrolase 1 of the maturepolypeptide of SEQ ID NO: 164. In another aspect, the Neosartoryafischeri Cel7 cellobiohydrolase 1 is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 163.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis an Aspergillus nidulans Cel7 cellobiohydrolase 1 of the maturepolypeptide of SEQ ID NO: 166. In another aspect, the Aspergillusnidulans Cel7 cellobiohydrolase 1 is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 165.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Myceliophthora thermophila Cel6A cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 10. In another aspect, the Myceliophthorathermophila Cel6A cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 9.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Myceliophthora thermophila Cel6B cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 12. In another aspect, the Myceliophthorathermophila Cel6B cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 11.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Thielavia terrestris Cel6A cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 14. In another aspect, the Thielaviaterrestris Cel6A cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 13.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Trichophaea saccata CBS 804.70 Cel6A cellobiohydrolase II of themature polypeptide of SEQ ID NO: 16. In another aspect, the Trichophaeasaccata Cel6A cellobiohydrolase II is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 15.

In another aspect, the polypeptide having cellobiohydrolase II activityis an Aspergillus fumigatus Cel6A cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 18. In another aspect, the Aspergillusfumigatus Cel6A cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 17.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Fennellia nivea Cel6 cellobiohydrolase II of the mature polypeptideof SEQ ID NO: 168. In another aspect, the Fennellia nivea Cel6cellobiohydrolase II is encoded by the mature polypeptide codingsequence of SEQ ID NO: 167.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Penicillium emersonii Cel6A cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 170. In another aspect, the Penicilliumemersonii Cel6A cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 169.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Penicillium pinophilum Cel6A cellobiohydrolase II of the maturepolypeptide of SEQ ID NO: 172. In another aspect, the Penicilliumpinophilum Cel6A cellobiohydrolase II is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 171.

In another aspect, the polypeptide having endoglucanase I activity is aAspergillus terreus Cel7A endoglucanase I of the mature polypeptide ofSEQ ID NO: 20. In another aspect, the Aspergillus terreus Cel7Aendoglucanase I is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 19.

In another aspect, the polypeptide having endoglucanase II activity is aTrichoderma reesei Cel5A endoglucanase II of the mature polypeptide ofSEQ ID NO: 22. In another aspect, the Trichoderma reesei Cel5Aendoglucanase II is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 21.

In another aspect, the polypeptide having endoglucanase II activity is aMyceliophthora thermophila Cel5A endoglucanase II of the maturepolypeptide of SEQ ID NO: 24. In another aspect, the Myceliophthorathermophila Cel5A endoglucanase II is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 23.

In another aspect, the polypeptide having endoglucanase II activity is aThermoascus aurantiacus Cel5A endoglucanase II of the mature polypeptideof SEQ ID NO: 26. In another aspect, the Thermoascus aurantiacus Cel5Aendoglucanase II is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 25.

In another aspect, the polypeptide having endoglucanase II activity isan Aspergillus fumigatus Cel5 endoglucanase II of the mature polypeptideof SEQ ID NO: 174. In another aspect, the Aspergillus fumigatus Cel5endoglucanase II is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 173.

In another aspect, the polypeptide having endoglucanase II activity is aNeosartorya fischeri Cel5 endoglucanase II of the mature polypeptide ofSEQ ID NO: 176. In another aspect, the Neosartorya fischeri Cel5endoglucanase II is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 175.

In another aspect, the polypeptide having beta-glucosidase activity is aAspergillus fumigatus beta-glucosidase of the mature polypeptide of SEQID NO: 28. In another aspect, the Aspergillus fumigatus beta-glucosidaseis encoded by the mature polypeptide coding sequence of SEQ ID NO: 27.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium brasilianum beta-glucosidase of the mature polypeptide ofSEQ ID NO: 30. In another aspect, the Penicillium brasilianumbeta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 29.

In another aspect, the polypeptide having beta-glucosidase activity is aAspergillus niger beta-glucosidase of the mature polypeptide of SEQ IDNO: 32. In another aspect, the Aspergillus niger beta-glucosidase isencoded by the mature polypeptide coding sequence of SEQ ID NO: 31.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus aculeatus Cel3 beta-glucosidase of the mature polypeptideof SEQ ID NO: 178. In another aspect, the Aspergillus aculeatus Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 177.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus kawashii Cel3 beta-glucosidase of the mature polypeptideof SEQ ID NO: 180. In another aspect, the Aspergillus kawashii Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 179.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus clavatus Cel3 beta-glucosidase of the mature polypeptideof SEQ ID NO: 182. In another aspect, the Aspergillus clavatus Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 181.

In another aspect, the polypeptide having beta-glucosidase activity is aThielavia terrestris NRRL 8126 Cel3 beta-glucosidase of the maturepolypeptide of SEQ ID NO: 184. In another aspect, the Thielaviaterrestris NRRL 8126 Cel3 beta-glucosidase is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 183.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium oxalicum Cel3 beta-glucosidase of the mature polypeptide ofSEQ ID NO: 186. In another aspect, the Penicillium oxalicum Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 185.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium oxalicum Cel3 beta-glucosidase of the mature polypeptide ofSEQ ID NO: 188. In another aspect, the Penicillium oxalicum Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 187.

In another aspect, the polypeptide having beta-glucosidase activity is aTalaromyces emersonii Cel3 beta-glucosidase of the mature polypeptide ofSEQ ID NO: 190. In another aspect, the Talaromyces emersonii Cel3beta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 189.

In one aspect, the enzyme composition further comprises or even furthercomprises a polypeptide having cellulolytic enhancing activity selectedfrom the group consisting of:

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 34; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 33, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 33, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 33;

(II) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 36; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 35, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 35, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 35;

(III) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 38; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 37, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 37, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 37;

(IV) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 40; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 39, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 39, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 39;

(V) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 42; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 41, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 41, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 41;

(VI) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 44; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 43, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 43, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 43;

(VII) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 192; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 191, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 191, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 191; and

(VIII) a combination of any of I, II, III, IV, V, VI, and VII.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity is a Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity of the mature polypeptide of SEQ ID NO:34. In another aspect, the Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 33.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thielavia terrestris GH61E polypeptide having cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 36. Inanother aspect, the Thielavia terrestris GH61E polypeptide havingcellulolytic enhancing activity is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 35.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity of the mature polypeptide of SEQ ID NO:38. In another aspect, the Aspergillus fumigatus GH61B polypeptidehaving cellulolytic enhancing activity is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 37.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Penicillium pinophilum GH61 polypeptide havingcellulolytic enhancing activity of the mature polypeptide of SEQ ID NO:40. In another aspect, the Penicillium pinophilum GH61A polypeptidehaving cellulolytic enhancing activity is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 39.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Penicillium sp. GH61A polypeptide having cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 42. Inanother aspect, the Penicillium sp. GH61 polypeptide having cellulolyticenhancing activity is encoded by the mature polypeptide coding sequenceof SEQ ID NO: 41.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thielavia terrestris GH61N polypeptide having cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 44. Inanother aspect, the Thielavia terrestris GH61N polypeptide havingcellulolytic enhancing activity is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 43.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thermoascus crustaceus GH61A polypeptide havingcellulolytic enhancing activity of the mature polypeptide of SEQ ID NO:192. In another aspect, the Thermoascus crustaceus GH61A polypeptidehaving cellulolytic enhancing activity is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 191.

In another aspect, the enzyme composition further comprises or evenfurther comprises a polypeptide having xylanase activity. In a preferredaspect, the polypeptide having xylanase activity is a Family 10polypeptide having xylanase activity. In another aspect, the polypeptidehaving xylanase activity is a Family 11 polypeptide having xylanaseactivity.

In a more preferred aspect, the Family 10 polypeptide having xylanaseactivity is selected from the group consisting of:

(I) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 46; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 45, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 45, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 45;

(II) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 48; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 47, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 47, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 47;

(III) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 50; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 49, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 49, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 49;

(IV) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 52; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 51, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 51, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 51;

(V) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 54; (b) a polypeptide encoded by a polynucleotide that hybridizesunder preferably at least medium-high stringency conditions, morepreferably at least high stringency conditions, and most preferably veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 53, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 53, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 53;

(VI) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 194; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 193, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 193, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 193;

(VII) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 196; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 195, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 195, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 195; and

(VIII) (a) a polypeptide comprising an amino acid sequence havingpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, even more preferably at least 95% identity, andmost preferably at least 97% identity to the mature polypeptide of SEQID NO: 198; (b) a polypeptide encoded by a polynucleotide thathybridizes under preferably at least medium-high stringency conditions,more preferably at least high stringency conditions, and most preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 197, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 197, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, even more preferably at least 95% identity, and most preferably atleast 97% identity to the mature polypeptide coding sequence of SEQ IDNO: 197.

In one aspect, the enzyme compositions comprise Aspergillus fumigatusCel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascus aurantiacusCel5A EGII, Penicillium sp GH61A polypeptide having cellulolyticenhancing activity, Aspergillus fumigatus GH3A beta-glucosidase,Aspergillus fumigatus GH10C xylanase, and Talaromyces emersonii GH3beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Penicillium sp GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B GH61 polypeptideshaving cellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise the enzymecompositions comprise Aspergillus fumigatus Cel 7A CBHI, Aspergillusfumigatus Cel 6A CBHII, Aspergillus fumigatus Cel5A EGII, Aspergillusfumigatus GH61B polypeptide having cellulolytic enhancing activity,Aspergillus fumigatus GH3A beta-glucosidase, Aspergillus fumigatus GH10Cxylanase, and Talaromyces emersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Aspergillus fumigatus GH61B polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Thermoascusaurantiacus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Aspergillusfumigatus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Penicilliumemersonii Cel 7 CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Aspergillus fumigatus GH10C xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Aspergillus fumigatus GH100 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus fumigatus GH3Abeta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

In another aspect, the enzyme compositions comprise Thermoascusaurantiacus Cel 7A CBHI, Aspergillus fumigatus Cel 6A CBHII, Aspergillusfumigatus Cel5A EGII, Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Aspergillus aculeatus GH3beta-glucosidase, Trichophaea saccata GH10 xylanase, and Talaromycesemersonii GH3 beta-xylosidase.

Enzyme Composition Components and Polynucleotides Thereof

In first aspect, the isolated polypeptides having cellobiohydrolase Iactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 2 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have cellobiohydrolase Iactivity (hereinafter “homologous polypeptides”). In a preferred aspect,the homologous polypeptides comprise amino acid sequences that differ byten amino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 2. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 2. In another aspect, the polypeptide comprises or consists of aminoacids 19 to 530 of SEQ ID NO: 2, or an allelic variant thereof; or afragment thereof having cellobiohydrolase I activity. In another aspect,the polypeptide comprises or consists of amino acids 19 to 530 of SEQ IDNO: 2.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 4 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In one aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:4.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 4. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 4. In another aspect, the polypeptide comprises or consists of aminoacids 21 to 450 of SEQ ID NO: 4, or an allelic variant thereof; or afragment thereof having cellobiohydrolase I activity. In another aspect,the polypeptide comprises or consists of amino acids 21 to 450 of SEQ IDNO: 4.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 6 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:6.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 6. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 6. In another aspect, the polypeptide comprises or consists of aminoacids 27 to 532 of SEQ ID NO: 6, or an allelic variant thereof; or afragment thereof having cellobiohydrolase I activity. In another aspect,the polypeptide comprises or consists of amino acids 27 to 532 of SEQ IDNO: 6.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 8 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:8.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 8 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 8. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 8. In another aspect, the polypeptide comprises or consists of aminoacids 18 to 457 of SEQ ID NO: 8, or an allelic variant thereof; or afragment thereof having cellobiohydrolase I activity. In another aspect,the polypeptide comprises or consists of amino acids 18 to 457 of SEQ IDNO: 8.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 158 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:158.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of or consists of the amino acid sequence of SEQID NO: 158 or an allelic variant thereof; or a fragment thereof havingcellobiohydrolase I activity. In another aspect, the polypeptidecomprises or consists of or consists of the amino acid sequence of SEQID NO: 158. In another aspect, the polypeptide comprises or consists ofor consists of the mature polypeptide of SEQ ID NO: 158. In anotheraspect, the polypeptide comprises or consists of or consists of aminoacids 19 to 455 of SEQ ID NO: 158, or an allelic variant thereof; or afragment thereof having cellobiohydrolase I activity. In another aspect,the polypeptide comprises or consists of or consists of amino acids 19to 455 of SEQ ID NO: 158.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 160 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:160.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 160 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 160. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 160. In another aspect, the polypeptide comprises or consists ofamino acids 26 to 529 of SEQ ID NO: 160, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase I activity. In anotheraspect, the polypeptide comprises or consists of amino acids 26 to 529of SEQ ID NO: 160.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 162 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:162.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 162 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 162. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 162. In another aspect, the polypeptide comprises or consists ofamino acids 24 to 541 of SEQ ID NO: 162, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase I activity. In anotheraspect, the polypeptide comprises or consists of amino acids 24 to 541of SEQ ID NO: 162.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 164 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:164.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 164 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 164. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 164. In another aspect, the polypeptide comprises or consists ofamino acids 27 to 535 of SEQ ID NO: 164, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase I activity. In anotheraspect, the polypeptide comprises or consists of amino acids 27 to 535of SEQ ID NO: 164.

In another first aspect, the isolated polypeptides havingcellobiohydrolase I activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 166 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase I activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:166.

In one aspect, a polypeptide having cellobiohydrolase I activitycomprises or consists of the amino acid sequence of SEQ ID NO: 166 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseI activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 166. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 166. In another aspect, the polypeptide comprises or consists ofamino acids 24 to 526 of SEQ ID NO: 166, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase I activity. In anotheraspect, the polypeptide comprises or consists of amino acids 24 to 526of SEQ ID NO: 166.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 10 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:10.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 10 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 10. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 10. In another aspect, the polypeptide comprises or consists ofamino acids 18 to 482 of SEQ ID NO: 10, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 482of SEQ ID NO: 10.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 12 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:12.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 12. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 12. In another aspect, the polypeptide comprises or consists ofamino acids 18 to 482 of SEQ ID NO: 12, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 482of SEQ ID NO: 12.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 14 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:14.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 14 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 14. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 14. In another aspect, the polypeptide comprises or consists ofamino acids 18 to 481 of SEQ ID NO: 14, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 481of SEQ ID NO: 14.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 16 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:16.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 16 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 16. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 16. In another aspect, the polypeptide comprises or consists ofamino acids 17 to 447 of SEQ ID NO: 16, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 17 to 447of SEQ ID NO: 16.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 18 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:18.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 18 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 18. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 18. In another aspect, the polypeptide comprises or consists ofamino acids 20 to 454 of SEQ ID NO: 18, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 454of SEQ ID NO: 18.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 168 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:168.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 168 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 168. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 168. In another aspect, the polypeptide comprises or consists ofamino acids 19 to 469 of SEQ ID NO: 168, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 19 to 469of SEQ ID NO: 168.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 170 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:170.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 170 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 170. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 170. In another aspect, the polypeptide comprises or consists ofamino acids 20 to 459 of SEQ ID NO: 170, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 459of SEQ ID NO: 170.

In another first aspect, the isolated polypeptides havingcellobiohydrolase II activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 172 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellobiohydrolase II activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:172.

In one aspect, a polypeptide having cellobiohydrolase II activitycomprises or consists of the amino acid sequence of SEQ ID NO: 172 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseII activity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 172. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 172. In another aspect, the polypeptide comprises or consists ofamino acids 20 to 457 of SEQ ID NO: 172, or an allelic variant thereof;or a fragment thereof having cellobiohydrolase II activity. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 457of SEQ ID NO: 172.

In another first aspect, the isolated polypeptides having endoglucanaseI activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 20 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase I activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 20.

In one aspect, a polypeptide having endoglucanase I activity comprisesor consists of the amino acid sequence of SEQ ID NO: 20 or an allelicvariant thereof; or a fragment thereof having endoglucanase I activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 20. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 20. Inanother aspect, the polypeptide comprises or consists of amino acids 22to 471 of SEQ ID NO: 20, or an allelic variant thereof; or a fragmentthereof having endoglucanase I activity. In another aspect, thepolypeptide comprises or consists of amino acids 22 to 471 of SEQ ID NO:20.

In another first aspect, the isolated polypeptides having endoglucanaseII activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 22 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase II activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 22.

In one aspect, a polypeptide having endoglucanase II activity comprisesor consists of the amino acid sequence of SEQ ID NO: 22 or an allelicvariant thereof; or a fragment thereof having endoglucanase II activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 22. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 22. Inanother aspect, the polypeptide comprises or consists of amino acids 22to 418 of SEQ ID NO: 22, or an allelic variant thereof; or a fragmentthereof having endoglucanase II activity. In another aspect, thepolypeptide comprises or consists of amino acids 22 to 418 of SEQ ID NO:22.

In another first aspect, the isolated polypeptides having endoglucanaseII activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 24 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase II activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 24.

In one aspect, a polypeptide having endoglucanase II activity comprisesor consists of the amino acid sequence of SEQ ID NO: 24 or an allelicvariant thereof; or a fragment thereof having endoglucanase II activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 24. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 24. Inanother aspect, the polypeptide comprises or consists of amino acids 17to 389 of SEQ ID NO: 24, or an allelic variant thereof; or a fragmentthereof having endoglucanase II activity. In another aspect, thepolypeptide comprises or consists of amino acids 17 to 389 of SEQ ID NO:24.

In another first aspect, the isolated polypeptides having endoglucanaseII activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 26 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase II activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 26.

In another first aspect, the isolated polypeptides having endoglucanaseII activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 174 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase II activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 174.

In one aspect, a polypeptide having endoglucanase II activity comprisesor consists of the amino acid sequence of SEQ ID NO: 174 or an allelicvariant thereof; or a fragment thereof having endoglucanase II activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 174. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 174. Inanother aspect, the polypeptide comprises or consists of amino acids 19to 329 of SEQ ID NO: 174, or an allelic variant thereof; or a fragmentthereof having endoglucanase II activity. In another aspect, thepolypeptide comprises or consists of amino acids 19 to 329 of SEQ ID NO:174.

In another first aspect, the isolated polypeptides having endoglucanaseII activity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 176 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have endoglucanase II activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 176.

In one aspect, a polypeptide having endoglucanase II activity comprisesor consists of the amino acid sequence of SEQ ID NO: 176 or an allelicvariant thereof; or a fragment thereof having endoglucanase II activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 176. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 176. Inanother aspect, the polypeptide comprises or consists of amino acids 17to 412 of SEQ ID NO: 176, or an allelic variant thereof; or a fragmentthereof having endoglucanase II activity. In another aspect, thepolypeptide comprises or consists of amino acids 17 to 412 of SEQ ID NO:176.

In one aspect, a polypeptide having endoglucanase II activity comprisesor consists of the amino acid sequence of SEQ ID NO: 26 or an allelicvariant thereof; or a fragment thereof having endoglucanase II activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 26. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 26. Inanother aspect, the polypeptide comprises or consists of amino acids 31to 335 of SEQ ID NO: 26, or an allelic variant thereof; or a fragmentthereof having endoglucanase II activity. In another aspect, thepolypeptide comprises or consists of amino acids 31 to 335 of SEQ ID NO:26.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 28 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:28.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 28 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 28. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 28. Inanother aspect, the polypeptide comprises or consists of amino acids 20to 863 of SEQ ID NO: 28, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 20 to 863 of SEQ ID NO:28.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 30 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:30.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 30 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 30. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 30. Inanother aspect, the polypeptide comprises or consists of amino acids 37to 878 of SEQ ID NO: 30, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 37 to 878 of SEQ ID NO:30.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 32 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:32.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 32 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 32. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 32. Inanother aspect, the polypeptide comprises or consists of amino acids 20to 860 of SEQ ID NO: 32, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 20 to 860 of SEQ ID NO:32.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 178 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:178.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 178 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 178. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 178. Inanother aspect, the polypeptide comprises or consists of amino acids 20to 680 of SEQ ID NO: 178, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 20 to 680 of SEQ ID NO:178.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 180 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:180.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 180 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 180. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 180. Inanother aspect, the polypeptide comprises or consists of amino acids 20to 860 of SEQ ID NO: 180, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 20 to 860 of SEQ ID NO:180.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 182 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:182.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 182 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 182. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 182. Inanother aspect, the polypeptide comprises or consists of amino acids 19to 860 of SEQ ID NO: 182, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 19 to 860 of SEQ ID NO:182.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 184 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:184.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 184 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 184. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 184. Inanother aspect, the polypeptide comprises or consists of amino acids 19to 872 of SEQ ID NO: 184, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 19 to 872 of SEQ ID NO:184.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 186 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:186.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 186 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 186. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 186. Inanother aspect, the polypeptide comprises or consists of amino acids 22to 883 of SEQ ID NO: 186, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 22 to 883 of SEQ ID NO:186.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 188 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:188.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 188 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 188. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 188. Inanother aspect, the polypeptide comprises or consists of amino acids 22to 861 of SEQ ID NO: 188, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 22 to 861 of SEQ ID NO:188.

In another first aspect, the isolated polypeptides havingbeta-glucosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 190 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:190.

In one aspect, a polypeptide having beta-glucosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 190 or an allelicvariant thereof; or a fragment thereof having beta-glucosidase activity.In another aspect, the polypeptide comprises or consists of the aminoacid sequence of SEQ ID NO: 190. In another aspect, the polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 190. Inanother aspect, the polypeptide comprises or consists of amino acids 22to 861 of SEQ ID NO: 190, or an allelic variant thereof; or a fragmentthereof having beta-glucosidase activity. In another aspect, thepolypeptide comprises or consists of amino acids 22 to 861 of SEQ ID NO:190.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 34 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:34.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 34 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 34. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 34. In another aspect, the polypeptide comprises or consists ofamino acids 23 to 250 of SEQ ID NO: 34, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 23 to 250of SEQ ID NO: 34.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 36 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:36.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 36 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 36. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 36. In another aspect, the polypeptide comprises or consists ofamino acids 20 to 258 of SEQ ID NO: 36, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 258of SEQ ID NO: 36.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 38 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:38.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 38 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 38. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 38. In another aspect, the polypeptide comprises or consists ofamino acids 22 to 250 of SEQ ID NO: 38, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 22 to 250of SEQ ID NO: 38.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 40 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:40.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 40 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 40. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 40. In another aspect, the polypeptide comprises or consists ofamino acids 22 to 322 of SEQ ID NO: 40, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 22 to 322of SEQ ID NO: 40.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 42 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:42.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 42 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 42. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 42. In another aspect, the polypeptide comprises or consists ofamino acids 26 to 253 of SEQ ID NO: 42, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 26 to 253of SEQ ID NO: 42.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 44 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:44.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 44 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 44. In another aspect,the polypeptide comprises or consists of the mature polypeptide of SEQID NO: 44. In another aspect, the polypeptide comprises or consists ofamino acids 22 to 368 of SEQ ID NO: 44, or an allelic variant thereof;or a fragment thereof having cellulolytic enhancing activity. In anotheraspect, the polypeptide comprises or consists of amino acids 22 to 368of SEQ ID NO: 44.

In another first aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity comprise amino acid sequences having adegree of identity to the mature polypeptide of SEQ ID NO: 192 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have cellulolytic enhancing activity (hereinafter “homologouspolypeptides”). In another aspect, the homologous polypeptides compriseamino acid sequences that differ by ten amino acids, preferably by fiveamino acids, more preferably by four amino acids, even more preferablyby three amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:192.

In one aspect, a polypeptide having cellulolytic enhancing activitycomprises or consists of the amino acid sequence of SEQ ID NO: 192 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 192. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 192. In another aspect, the polypeptide comprises orconsists of amino acids 23 to 251 of SEQ ID NO: 186, or an allelicvariant thereof; or a fragment thereof having cellulolytic enhancingactivity. In another aspect, the polypeptide comprises or consists ofamino acids 23 to 251 of SEQ ID NO: 186.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 46, of at preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 46.

In one aspect, a GH10 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 46 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 46. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 46. In anotheraspect, the polypeptide comprises or consists of amino acids 23 to 406of SEQ ID NO: 46, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 23 to 406 of SEQ ID NO: 46.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 48, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 48.

In one aspect, a GH10 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 48 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 48. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 48. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 397of SEQ ID NO: 48, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 20 to 397 of SEQ ID NO: 48.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 50, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 50.

In one aspect, a GH10 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 50 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 50. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 50. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 398of SEQ ID NO: 50, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 20 to 398 of SEQ ID NO: 50.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 52, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 52.

In one aspect, a GH10 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 50 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 52. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 52. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 407of SEQ ID NO: 52, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 20 to 407 of SEQ ID NO: 52.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 54, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 54.

In one aspect, a GH10 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 50 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 54. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 54. In anotheraspect, the polypeptide comprises or consists of amino acids 20 to 395of SEQ ID NO: 54, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 20 to 395 of SEQ ID NO: 54.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 194, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 194.

A GH10 polypeptide having xylanase activity comprises or consists of theamino acid sequence of SEQ ID NO: 194 or an allelic variant thereof; ora fragment thereof that has xylanase activity. In another aspect, thepolypeptide comprises or consists of the amino acid sequence of SEQ IDNO: 194. In another aspect, the polypeptide comprises or consists of themature polypeptide of SEQ ID NO: 194. In another aspect, the polypeptidecomprises or consists of amino acids 24 to 403 of SEQ ID NO: 194, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another aspect, the polypeptide comprises or consists ofamino acids 24 to 403 of SEQ ID NO: 194.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 196, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 196.

A GH10 polypeptide having xylanase activity comprises or consists of theamino acid sequence of SEQ ID NO: 194 or an allelic variant thereof; ora fragment thereof that has xylanase activity. In another aspect, thepolypeptide comprises or consists of the amino acid sequence of SEQ IDNO: 196. In another aspect, the polypeptide comprises or consists of themature polypeptide of SEQ ID NO: 196. In another aspect, the polypeptidecomprises or consists of amino acids 24 to 403 of SEQ ID NO: 196, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another aspect, the polypeptide comprises or consists ofamino acids 24 to 403 of SEQ ID NO: 196.

In another first aspect, the isolated GH10 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 198, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 198.

A GH10 polypeptide having xylanase activity comprises or consists of theamino acid sequence of SEQ ID NO: 194 or an allelic variant thereof; ora fragment thereof that has xylanase activity. In another aspect, thepolypeptide comprises or consists of the amino acid sequence of SEQ IDNO: 198. In another aspect, the polypeptide comprises or consists of themature polypeptide of SEQ ID NO: 198. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 396 of SEQ ID NO: 198, or anallelic variant thereof; or a fragment thereof that has xylanaseactivity. In another aspect, the polypeptide comprises or consists ofamino acids 20 to 396 of SEQ ID NO: 198.

In another first aspect, the isolated GH11 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 56, of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have xylanase activity(hereinafter “homologous polypeptides”). In another aspect, thehomologous polypeptides comprise amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 56.

In one aspect, a GH11 polypeptide having xylanase activity comprises orconsists of the amino acid sequence of SEQ ID NO: 56 or an allelicvariant thereof; or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of the amino acidsequence of SEQ ID NO: 56. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 56. In anotheraspect, the polypeptide comprises or consists of amino acids 43 to 338of SEQ ID NO: 56, or an allelic variant thereof; or a fragment thereofthat has xylanase activity. In another aspect, the polypeptide comprisesor consists of amino acids 43 to 338 of SEQ ID NO: 56. In anotheraspect, the polypeptide comprises or consists of amino acids 43 to 338of SEQ ID NO: 56. In another aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 56 or an allelic variant thereof; or afragment thereof that has xylanase activity.

In another first aspect, the isolated GH11 polypeptides having xylanaseactivity comprise amino acid sequences having a degree of identity tothe mature polypeptide of SEQ ID NO: 200 or SEQ ID NO: 305, ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which have xylanase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:200 or SEQ ID NO: 305.

A GH11 polypeptide having xylanase activity comprises or consists of theamino acid sequence of SEQ ID NO: 200, or an allelic variant thereof; ora fragment thereof that has xylanase activity, or SEQ ID NO: 305 or afragment thereof that has xylanase activity. In another aspect, thepolypeptide comprises or consists of the amino acid sequence of SEQ IDNO: 200 or SEQ ID NO: 305. In another aspect, the polypeptide comprisesor consists of the mature polypeptide of SEQ ID NO: 200 or SEQ ID NO:305. In another aspect, the polypeptide comprises or consists of aminoacids 25 to 360 of SEQ ID NO: 200, or an allelic variant thereof; or afragment thereof that has xylanase activity, or amino acids 29 to 231 ofSEQ ID NO: 305 or a fragment thereof that has xylanase activity. Inanother aspect, the polypeptide comprises or consists of amino acids 25to 360 of SEQ ID NO: 200 or amino acids 29 to 231 of SEQ ID NO: 305. Inanother aspect, the polypeptide comprises or consists of amino acids 25to 360 of SEQ ID NO: 200 or amino acids 29 to 231 of SEQ ID NO: 305.

In another first aspect, the isolated polypeptides havingbeta-xylosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 58, of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-xylosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:58.

In one aspect, a polypeptide having beta-xylosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 58 or an allelicvariant thereof; or a fragment thereof that has beta-xylosidaseactivity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 58. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 58. In another aspect, the polypeptide comprises or consists ofamino acids 21 to 797 of SEQ ID NO: 58, or an allelic variant thereof;or a fragment thereof that has beta-xylosidase activity. In anotheraspect, the polypeptide comprises or consists of amino acids 21 to 797of SEQ ID NO: 58.

In another first aspect, the isolated polypeptides havingbeta-xylosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 60, of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which havebeta-xylosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:60.

In one aspect, a polypeptide having beta-xylosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 60 or an allelicvariant thereof; or a fragment thereof that has beta-xylosidaseactivity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 60. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 60. In another aspect, the polypeptide comprises or consists ofamino acids 22 to 795 of SEQ ID NO: 60, or an allelic variant thereof;or a fragment thereof that has beta-xylosidase activity. In anotheraspect, the polypeptide comprises or consists of amino acids 22 to 795of SEQ ID NO: 60.

In another first aspect, the isolated polypeptides havingbeta-xylosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 202, of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which havebeta-xylosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:202.

In one aspect, a polypeptide having beta-xylosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 202 or an allelicvariant thereof; or a fragment thereof that has beta-xylosidaseactivity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 202. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 202. In another aspect, the polypeptide comprises or consists ofamino acids 18 to 803 of SEQ ID NO: 202, or an allelic variant thereof;or a fragment thereof that has beta-xylosidase activity. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 803of SEQ ID NO: 202.

In one aspect, a polypeptide having beta-xylosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 204 or an allelicvariant thereof; or a fragment thereof that has beta-xylosidaseactivity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 204. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 204. In another aspect, the polypeptide comprises or consists ofamino acids 18 to 817 of SEQ ID NO: 204, or an allelic variant thereof;or a fragment thereof that has beta-xylosidase activity. In anotheraspect, the polypeptide comprises or consists of amino acids 18 to 817of SEQ ID NO: 204.

In another first aspect, the isolated polypeptides havingbeta-xylosidase activity comprise amino acid sequences having a degreeof identity to the mature polypeptide of SEQ ID NO: 206, of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which havebeta-xylosidase activity (hereinafter “homologous polypeptides”). Inanother aspect, the homologous polypeptides comprise amino acidsequences that differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:206.

In one aspect, a polypeptide having beta-xylosidase activity comprisesor consists of the amino acid sequence of SEQ ID NO: 206 or an allelicvariant thereof; or a fragment thereof that has beta-xylosidaseactivity. In another aspect, the polypeptide comprises or consists ofthe amino acid sequence of SEQ ID NO: 206. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 206. In another aspect, the polypeptide comprises or consists ofamino acids 21 to 792 of SEQ ID NO: 206, or an allelic variant thereof;or a fragment thereof that has beta-xylosidase activity. In anotheraspect, the polypeptide comprises or consists of amino acids 21 to 792of SEQ ID NO: 206.

In a second aspect, the isolated polypeptides having cellobiohydrolase Iactivity are encoded by polynucleotides that hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomicDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1,or (iii) a full-length complementary strand of (i) or (ii) (J. Sambrook,E. F. Fritsch, and T. Maniatis, 1989, supra).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 3, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 3, or (iii) a full-length complementarystrand of (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 5, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 5, or (iii) a full-length complementary strand of(i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 7, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 7, or (iii) a full-length complementarystrand of (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 157, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 157, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 159, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 159, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 161, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 161, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 163, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 163, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 165, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 165, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 9, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 9, or (iii) a full-length complementary strand of(i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 11, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 11, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 13, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 13, or (iii) a full-length complementarystrand of (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 15, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 15, or (iii) a full-length complementarystrand of (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 17, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 17, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 167, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 167, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 169, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 169, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 171, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 171, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides having endoglucanaseI activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:19, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 19, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:21, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 21, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:23, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 23, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:25, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 25, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:173, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 173, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides that hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:175, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 175, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:27, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 27, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:29, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 29, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:31, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 31, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:177, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 177, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:179, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 179, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:181, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 181, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:183, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 183, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:185, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 185, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:187, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 187, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides that hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:189, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 189, or (iii) a full-length complementary strand of (i) or(ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 33, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 33, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 35, (ii) the genomic DNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 35, or (iii) a full-length complementarystrand of (i) or (ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 37, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 37, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 39, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 39, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 41, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 41, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotides thathybridize under preferably medium-high stringency conditions, morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 43, (ii) the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 43, or (iii) a full-length complementary strandof (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 191, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 191, or (iii) afull-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity are encoded by polynucleotides that hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 45, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 45, or (iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity are encoded by polynucleotides that hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 47, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 47, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 49, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 49, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 51, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 51, or (iii) afull-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 53, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 53, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 193, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 193, or (iii) afull-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 195, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 195, or (iii) afull-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH10 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 197, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 197, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated GH11 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with the mature polypeptidecoding sequence of SEQ ID NO: 55 or its full-length complementarystrand.

In another second aspect, the isolated GH11 polypeptides having xylanaseactivity is encoded by polynucleotides that hybridize under preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with the mature polypeptidecoding sequence of SEQ ID NO: 199 or SEQ ID NO: 304; or its full-lengthcomplementary strand.

In another second aspect, the isolated polypeptides havingbeta-xylosidase activity is encoded by polynucleotides that hybridizeunder preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 57, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 57, or (iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated polypeptides havingbeta-xylosidase activity is encoded by polynucleotides that hybridizeunder preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 59, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 59, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated polypeptides havingbeta-xylosidase activity is encoded by polynucleotides that hybridizeunder preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 201, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 201, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated polypeptides havingbeta-xylosidase activity is encoded by polynucleotides that hybridizeunder preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 203, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 203, or(iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the isolated polypeptides havingbeta-xylosidase activity is encoded by polynucleotides that hybridizeunder preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 205, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 205, or(iii) a full-length complementary strand of (i) or (ii).

The nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 919, 193, 195, 197, 199, 201, 203, or 205; or asubsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, or 206; or a fragment thereof; may be used to designnucleic acid probes to identify and clone DNA encoding polypeptideshaving enzyme activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength. For example, the nucleic acid probe may be at least 200nucleotides, preferably at least 300 nucleotides, more preferably atleast 400 nucleotides, or most preferably at least 500 nucleotides inlength. Even longer probes may be used, e.g., nucleic acid probes thatare preferably at least 600 nucleotides, more preferably at least 700nucleotides, even more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having enzyme or biological activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 919, 193, 195,197, 199, 201, 203, or 205 or a subsequence thereof, the carriermaterial is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 919, 193, 195,197, 199, 201, 203, or 205; the cDNA sequence of or the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 919, 193, 195, 197,199, 201, 203, or 205; its full-length complementary strand; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 55 to 1590 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide sequence that encodes the polypeptide ofSEQ ID NO: 2, or a subsequence thereof. In another aspect, the nucleicacid probe is SEQ ID NO: 1. In another aspect, the nucleic acid probe isthe polynucleotide sequence contained in Chaetomium thermophilum CGMCC0581, wherein the polynucleotide sequence thereof encodes a polypeptidehaving cellobiohydrolase I activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in Chaetomiumthermophilum CGMCC 0581.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 3. In another aspect, the nucleic acidprobe is nucleotides 61 to 1350 of SEQ ID NO: 3. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 4, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 3. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inMyceliophthora thermophila CBS 117.65, wherein the polynucleotidesequence thereof encodes a polypeptide having cellobiohydrolase Iactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in Myceliophthora thermophila CBS117.65.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 5. In another aspect, the nucleic acidprobe is nucleotides 79 to 1596 of SEQ ID NO: 5. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 6, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 5. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus fumigatus NN055679, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus fumigatus NN055679.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 7. In another aspect, the nucleic acidprobe is nucleotides 52 to 1374 of SEQ ID NO: 7. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 8, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 7. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inThermoascus aurantiacus CGMCC 0583, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Thermoascus aurantiacus CGMCC 0583.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 157. In another aspect, the nucleic acidprobe is nucleotides 55 to 1428 of SEQ ID NO: 157. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 158, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 157. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillium emersonii NN051602, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Penicillium emersonii NN051602.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 159. In another aspect, the nucleic acidprobe is nucleotides 76 to 1590 of SEQ ID NO: 159. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 160, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 159. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillium pinophilum NN046877, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Penicillium pinophilum NN046877.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 161. In another aspect, the nucleic acidprobe is nucleotides 52 to 1374 of SEQ ID NO: 161. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 162, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 161. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus terreus ATCC 28865, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus terreus ATCC 28865.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 163. In another aspect, the nucleic acidprobe is nucleotides 79 to 1605 of SEQ ID NO: 163. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 164, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 162. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inNeosartorya fischeri NRRL 181, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Neosartorya fischeri NRRL 181.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 165. In another aspect, the nucleic acidprobe is nucleotides 52 to 1374 of SEQ ID NO: 165. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 166, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 165. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus nidulans FGSCA4, wherein the polynucleotide sequence thereofencodes a polypeptide having cellobiohydrolase I activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Aspergillus nidulans FGSCA4.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 9. In another aspect, the nucleic acidprobe is nucleotides 52 to 1799 of SEQ ID NO: 9. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 10, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 9. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inMyceliophthora thermophila CBS 117.65, wherein the polynucleotidesequence thereof encodes a polypeptide having cellobiohydrolase IIactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in Myceliophthora thermophila CBS117.65.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 11. In another aspect, the nucleic acidprobe is nucleotides 52 to 1809 of SEQ ID NO: 11. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 12, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 11. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpSMai182 which is contained in E. coli NRRL B-50059 or contained inMyceliophthora thermophila CBS 202.73, wherein the polynucleotidesequence thereof encodes a polypeptide having cellobiohydrolase IIactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pSMai182 which iscontained in E. coli NRRL B-50059 or contained in Myceliophthorathermophila CBS 202.73.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 13. In another aspect, the nucleic acidprobe is nucleotides 52 to 1443 of SEQ ID NO: 13. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 14, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 13. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpTter6A which is contained in E. coli NRRL B-30802, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellobiohydrolase II activity. In another aspect, the nucleic acid probeis the mature polypeptide coding region contained in plasmid pTter6Awhich is contained in E. coli NRRL B-30802.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 15. In another aspect, the nucleic acidprobe is nucleotides 109 to 1401 of SEQ ID NO: 15. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 16, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 15. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpMStr199 which is contained in E. coli DSM 23379, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellobiohydrolase II activity. In another aspect, the nucleic acid probeis the mature polypeptide coding region contained in plasmid pMStr199which is contained in E. coli DSM 23379.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 17. In another aspect, the nucleic acidprobe is nucleotides 58 to 1700 of SEQ ID NO: 17. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 18, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 17. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus fumigatus NN055679, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase II activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus fumigatus NN055679.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 167. In another aspect, the nucleic acidprobe is nucleotides 55 to 1749 of SEQ ID NO: 167. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 168, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 167. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in Fennellianivea NN046949 or in pGEM-T-CBHII46949-2 which is contained in E. coliDSM 24143, wherein the polynucleotide sequence thereof encodes apolypeptide having cellobiohydrolase 11 activity. In another aspect, thenucleic acid probe is the mature polypeptide coding region contained inFennellia nivea NN046949 or in pGEM-T-CBHII46949-2 which is contained inE. coli DSM 24143.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 169. In another aspect, the nucleic acidprobe is nucleotides 58 to 1744 of SEQ ID NO: 169. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 170, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 169. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillium emersonii NN051602, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase 11 activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Penicillium emersonii NN051602.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 171. In another aspect, the nucleic acidprobe is nucleotides 58 to 1701 of SEQ ID NO: 171. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 172, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 171. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillium pinophilum NN046877, wherein the polynucleotide sequencethereof encodes a polypeptide having cellobiohydrolase II activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Penicillium pinophilum NN046877.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 19. In another aspect, the nucleic acidprobe is nucleotides 64 to 1502 of SEQ ID NO: 19. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 20, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 19. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus terreus ATCC 28865, wherein the polynucleotide sequencethereof encodes a polypeptide having endoglucanase I activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus terreus ATCC 28865.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 21. In another aspect, the nucleic acidprobe is nucleotides 64 to 1254 of SEQ ID NO: 21. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 22, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 21. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inTrichoderma reesei RutC30, wherein the polynucleotide sequence thereofencodes a polypeptide having endoglucanase II activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Trichoderma reesei RutC30.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 23. In another aspect, the nucleic acidprobe is nucleotides 67 to 1185 of SEQ ID NO: 23. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 24, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 23. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpCIC161 which is contained in E. coli NRRL B-30902, wherein thepolynucleotide sequence thereof encodes a polypeptide havingendoglucanase II activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in plasmid pCIC161 whichis contained in E. coli NRRL B-30902.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 25. In another aspect, the nucleic acidprobe is nucleotides 91 to 1005 of SEQ ID NO: 25. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 26, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 25. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inThermoascus aurantiacus CGMCC 0670, wherein the polynucleotide sequencethereof encodes a polypeptide having endoglucanase II activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Thermoascus aurantiacus CGMCC 0670.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 173. In another aspect, the nucleic acidprobe is nucleotides 55 to 1260 of SEQ ID NO: 173. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 174, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 173. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus fumigatus NN051616, wherein the polynucleotide sequencethereof encodes a polypeptide having endoglucanase II activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus fumigatus NN051616.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 175. In another aspect, the nucleic acidprobe is nucleotides 49 to 1378 of SEQ ID NO: 175. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 176, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 175. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inNeosartorya fischeri NRRL 181, wherein the polynucleotide sequencethereof encodes a polypeptide having endoglucanase II activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Neosartorya fischeri NRRL 181.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 27. In another aspect, the nucleic acidprobe is nucleotides 58 to 2580 of SEQ ID NO: 27. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 28, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 27. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpEJG113 which is contained in E. coli NRRL B-30695, wherein thepolynucleotide sequence thereof encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in plasmid pEJG113 whichis contained in E. coli NRRL B-30695.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 29. In another aspect, the nucleic acidprobe is nucleotides 171 to 2753 of SEQ ID NO: 29. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 30, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 29. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpKKAB which is contained in E. coli NRRL B-30860, wherein thepolynucleotide sequence thereof encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in plasmid pKKAB which iscontained in E. coli NRRL B-30860.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 31. In another aspect, the nucleic acidprobe is nucleotides 58 to 2934 of SEQ ID NO: 31. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 32, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 31. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus niger IBT 10140, wherein the polynucleotide sequence thereofencodes a polypeptide having beta-glucosidase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Aspergillus niger IBT 10140.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 177. In another aspect, the nucleic acidprobe is nucleotides 58 to 2937 of SEQ ID NO: 177. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 178, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 177. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus aculeatus WDCM190, which encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in Aspergillus aculeatusWDCM190.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 179. In another aspect, the nucleic acidprobe is nucleotides 58 to 2932 of SEQ ID NO: 179. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 180, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 179. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus kawashii IFO4308, which encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in Aspergillus kawashiiIFO4308.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 181. In another aspect, the nucleic acidprobe is nucleotides 55 to 3059 of SEQ ID NO: 181. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 182, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 181. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus clavatus NRRL 1, which encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in Aspergillus clavatusNRRL 1.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 183. In another aspect, the nucleic acidprobe is nucleotides 55 to 3029 of SEQ ID NO: 183. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 184, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 183. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in Thielaviaterrestris NRRL 8126, which encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in Thielavia terrestrisNRRL 8126.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 185. In another aspect, the nucleic acidprobe is nucleotides 64 to 2790 of SEQ ID NO: 185. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 186, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 185. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in pUC19D55EX which is contained in E. coli NRRL B-50395, wherein thepolynucleotide sequence thereof encodes a polypeptide havingbeta-glucosidase activity. In another aspect, the nucleic acid probe isthe mature polypeptide coding region contained in in pUC19 D55EX whichis contained in E. coli NRRL B-50395.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 187. In another aspect, the nucleic acidprobe is nucleotides 64 to 2790 of SEQ ID NO: 187. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 188, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 187. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillium oxalicum IBT5387, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-glucosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Penicillium oxalicum IBT5387.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 189. In another aspect, the nucleic acidprobe is nucleotides 58 to 2961 of SEQ ID NO: 189. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 190, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 189. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inTalaromyces emersonii CBS 549.92, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-glucosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Talaromyces emersonii CBS 549.92.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 33. In another aspect, the nucleic acidprobe is nucleotides 67 to 796 of SEQ ID NO: 33. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 34, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 33. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpDZA2-7 which is contained in E. coli NRRL B-30704, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpDZA2-7 which is contained in E. coli NRRL B-30704.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 35. In another aspect, the nucleic acidprobe is nucleotides 58 to 900 of SEQ ID NO: 35. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 36, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 35. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpTter61E which is contained in E. coli NRRL B-30814, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpTter61E which is contained in E. coli NRRL B-30814.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 37. In another aspect, the nucleic acidprobe is nucleotides 64 to 859 of SEQ ID NO: 37. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 38, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 37. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus fumigatus NN051616, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus fumigatus NN051616.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 39. In another aspect, the nucleic acidprobe is nucleotides 64 to 1018 of SEQ ID NO: 39. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 40, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 39. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inPenicillim pinophilum NN046877, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in plasmid pGEM-T-Ppin7 which is contained in E. coliDSM 22711.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 41. In another aspect, the nucleic acidprobe is nucleotides 76 to 832 of SEQ ID NO: 41. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 42, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 41. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 43. In another aspect, the nucleic acidprobe is nucleotides 64 to 1104 of SEQ ID NO: 43. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 44, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 43. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpAG68 which is contained in E. coli NRRL B-50320, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmid pAG68which is contained in E. coli NRRL B-50320.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 191. In another aspect, the nucleic acidprobe is nucleotides 64 to 1104 of SEQ ID NO: 191. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 192, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 191. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpGEM-T-GH61a51486 which is contained in E. coli DSM 22656, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another aspect, the nucleic acidprobe is the mature polypeptide coding region contained in plasmidpGEM-T-GH61a51486 which is contained in E. coli DSM 22656.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 45. In another aspect, the nucleic acidprobe is nucleotides 69 to 1314 of SEQ ID NO: 45. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 46, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 45. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergilluε aculeatus CBS 101.43, wherein the polynucleotide sequencethereof encodes a polypeptide having xylanase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Aspergilluε aculeatus CBS 101,43.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 47. In another aspect, the nucleic acidprobe is nucleotides 107 to 1415 of SEQ ID NO: 47. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 48, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 47. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpHyGe001 which is contained in E. coli NRRL B-30703, wherein thepolynucleotide sequence thereof encodes a polypeptide having xylanaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pHyGe001 which iscontained in E. coli NRRL B-30703.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 49. In another aspect, the nucleic acidprobe is nucleotides 58 to 1194 of SEQ ID NO: 49. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 50, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 49. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpTF12Xy1170 which is contained in E. coli NRRL B-50309, wherein thepolynucleotide sequence thereof encodes a polypeptide having xylanaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pTF12Xy1170 which iscontained in E. coli NRRL B-50309.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 51. In another aspect, the nucleic acidprobe is nucleotides 58 to 1439 of SEQ ID NO: 51. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 52, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 51. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in plasmidpGEM-T-Ppin3 which is contained in E. coli DSM 22922, wherein thepolynucleotide sequence thereof encodes a polypeptide having xylanaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pGEM-T-Ppin3 which iscontained in E. coli DSM 22922.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 53. In another aspect, the nucleic acidprobe is nucleotides 58 to 1185 of SEQ ID NO: 53. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 54, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 53. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in Thielaviaterrestris NRRL 8126, wherein the polynucleotide sequence thereofencodes a polypeptide having xylanase activity. In another aspect, thenucleic acid probe is the mature polypeptide coding region contained inThielavia terrestris NRRL 8126.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 193. In another aspect, the nucleic acidprobe is nucleotides 70 to 1383 of SEQ ID NO: 193. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 194, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 193. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inTalaromyces emersonii NN050022, wherein the polynucleotide sequencethereof encodes a polypeptide having xylanase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Talaromyces emersonii NN050022.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 195. In another aspect, the nucleic acidprobe is nucleotides 70 to 1384 of SEQ ID NO: 195. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 196, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 195. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in pMMar26which is contained in E. coli NRRL B-50266, wherein the polynucleotidesequence thereof encodes a polypeptide having xylanase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in pMMar26 which is contained in E. coli NRRL B-50266.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 197. In another aspect, the nucleic acidprobe is nucleotides 58 to 1188 of SEQ ID NO: 197. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 198, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 197. In another aspect, thenucleic acid probe is the polynucleotide sequence contained in E. coliDSM 10361, wherein the polynucleotide sequence thereof encodes apolypeptide having xylanase activity. In another aspect, the nucleicacid probe is the mature polypeptide coding region contained in E. coliDSM 10361.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 55. In another aspect, the nucleic acidprobe is nucleotides 127 to 1014 of SEQ ID NO: 55. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 56, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 55. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inThermobifida fusca DSM 22883, wherein the polynucleotide sequencethereof encodes a polypeptide having xylanase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Thermobifida fusca DSM 22883.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 55. In another aspect, the nucleic acidprobe is nucleotides 85 to 693 of SEQ ID NO: 55. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 56, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 55. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inDictyoglomus thermophilum ATCC 35947, wherein the polynucleotidesequence thereof encodes a polypeptide having xylanase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Dictyoglomus thermophilum ATCC 35947.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 57. In another aspect, the nucleic acidprobe is nucleotides 61 to 2391 of SEQ ID NO: 57. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 58, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 57. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inTrichoderma reesei RutC30, wherein the polynucleotide sequence thereofencodes a polypeptide having beta-xylosidase activity. In anotheraspect, the nucleic acid probe is the mature polypeptide coding regioncontained in Trichoderma reesei RutC30.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 59. In another aspect, the nucleic acidprobe is nucleotides 64 to 2388 of SEQ ID NO: 59. In another aspect, thenucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 60, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 59. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inTalaromyces emersonii CBS 393.64, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-xylosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Talaromyces emersonii CBS 393.64.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 201. In another aspect, the nucleic acidprobe is nucleotides 52 to 2409 of SEQ ID NO: 201. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 202, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 201. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus aculeatus CBS 172.66, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-xylosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus aculeatus CBS 172.66.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 203. In another aspect, the nucleic acidprobe is nucleotides 52 to 2451 of SEQ ID NO: 203. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 204, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 203. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus aculeatus CBS 186.67, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-xylosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus aculeatus CBS 186.67.

In another aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 205. In another aspect, the nucleic acidprobe is nucleotides 61 to 2376 of SEQ ID NO: 205. In another aspect,the nucleic acid probe is a polynucleotide sequence that encodes thepolypeptide of SEQ ID NO: 206, or a subsequence thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 205. In another aspect, thenucleic acid probe is the polynucleotide sequence contained inAspergillus fumigatus NN051616, wherein the polynucleotide sequencethereof encodes a polypeptide having beta-xylosidase activity. Inanother aspect, the nucleic acid probe is the mature polypeptide codingregion contained in Aspergillus fumigatus NN051616.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2×SSC, 0.2% SDS at 45° C. (very low stringency), at 50°C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

In a third aspect, the isolated polypeptides having cellobiohydrolase Iactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 3 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 5 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 7 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 157 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 159 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 161 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 163 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase I activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 165 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase I activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 9 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 11 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 13 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 15 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 17 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 167 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 169 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides havingcellobiohydrolase II activity are encoded by polynucleotides comprisingor consisting of nucleotide sequences that have a degree of identity tothe mature polypeptide coding sequence of SEQ ID NO: 171 of preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having cellobiohydrolase II activity.

In another third aspect, the isolated polypeptides having endoglucanaseI activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 19 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase I activity.

In another third aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 21 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In another third aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 23 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In another third aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 25 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In another third aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 173 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving endoglucanase II activity.

In another third aspect, the isolated polypeptides having endoglucanaseII activity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 175 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving endoglucanase II activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 27 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 29 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 31 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 177 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 179 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 181 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 183 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 185 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 187 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated polypeptides havingbeta-glucosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 189 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-glucosidase activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 33 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 35 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 37 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 39 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 41 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 43 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated GH61 polypeptides havingcellulolytic enhancing activity are encoded by polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 191 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having cellulolytic enhancing activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 45 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 47 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 49 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 51 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 53 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 193 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving xylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 195 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving xylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 197 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving xylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 55 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In another third aspect, the isolated polypeptides having xylanaseactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 199 or SEQ ID NO: 304 ofpreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having xylanase activity.

In another third aspect, the isolated polypeptides havingbeta-xylosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 57 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-xylosidase activity.

In another third aspect, the isolated polypeptides havingbeta-xylosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 59 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-xylosidase activity.

In another third aspect, the isolated polypeptides havingbeta-xylosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 201 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-xylosidase activity.

In another third aspect, the isolated polypeptides havingbeta-xylosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 203 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-xylosidase activity.

In another third aspect, the isolated polypeptides havingbeta-xylosidase activity are encoded by polynucleotides comprising orconsisting of nucleotide sequences that have a degree of identity to themature polypeptide coding sequence of SEQ ID NO: 205 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having beta-xylosidase activity.

Techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of apolynucleotide from such genomic DNA can be effected, e.g., by using thewell known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligatedactivated transcription (LAT) and nucleotide sequence-basedamplification (NASBA) may be used. The polynucleotides may be clonedfrom any strain and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleotide sequence.

In a fourth aspect, the isolated polynucleotides encoding polypeptideshaving cellobiohydrolase I activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 1. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Chaetomiumthermophilum CGMCC 0581. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 1. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 55 to 1590 of SEQ ID NO: 1. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Chaetomium thermophilum CGMCC 0581.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 3 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 3. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Myceliophthorathermophila CBS 117.65. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 3. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 61 to 1350 of SEQ ID NO: 3. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Myceliophthora thermophila CBS 117.65.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 5 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 5. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus fumigatusNN055679. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 5. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 79 to 1596 of SEQ ID NO: 5. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus fumigatus NN055679.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 7 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 7. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Thermoascusaurantiacus CGMCC 0583. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 7. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 52 to 1374 of SEQ ID NO: 7. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Thermoascus aurantiacus CGMCC 0583.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 157 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 157. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Penicillium emersoniiNN051602. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 157. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 55 to 1428 of SEQ ID NO: 157. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Penicillium emersonii NN051602

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 159 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 159. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Penicilliumpinophilum NN046877. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 159. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 76 to 1590 of SEQ ID NO: 159. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Penicillium pinophilum NN046877.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 161 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 161. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus terreusATCC 28865. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 161. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 70 to 1675 of SEQ ID NO: 161. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus terreus ATCC 28865.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 163 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 163. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Neosartorya fischeriNRRL 181. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 163. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 79 to 1605 of SEQ ID NO: 163. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Neosartorya fischeri NRRL 181.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 165 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 165. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus nidulansstrain FGSCA4. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 165. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 70 to 1578 of SEQ ID NO: 165. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus nidulans strain FGSCA4.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 9 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 9. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Myceliophthorathermophila CBS 117.65. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 9. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 52 to 1799 of SEQ ID NO: 9. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Myceliophthora thermophila CBS 117.65.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 11 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 11. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pSMai182which is contained in E. coli NRRL B-50059 or contained inMyceliophthora thermophila CBS 202.73. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 11. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 52 to 1809 of SEQ ID NO: 11. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in Myceliophthora thermophila CBS202.73 or contained in plasmid pSMai182 which is contained in E. coliNRRL B-50059.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 13 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 13. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pTter6A whichis contained in E. coli NRRL B-30802. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 13. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 52 to 1443 of SEQ ID NO: 13. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pTter6A which iscontained in E. coli NRRL B-30802.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 15 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 15. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pMStr199which is contained in E. coli DSM 23379. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 15. In another aspect, the nucleotidesequence comprises or consists of nucleotides 109 to 1401 of SEQ ID NO:15. In another aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pMStr199which is contained in E. coli DSM 23379.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 17 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 17. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus fumigatusNN055679. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 17. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 58 to 1700 of SEQ ID NO: 17. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus fumigatus NN055679.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 167 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 167. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in pGEM-T-CBHII46949-2which is contained in E. coli DSM 24143. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 167. In another aspect, the nucleotidesequence comprises or consists of nucleotides 55 to 1749 of SEQ ID NO:167. In another aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in pGEM-T-CBHII46949-2which is contained in E. coli DSM 24143.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 169 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 169. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Penicillium emersoniiNN051602. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 169. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 58 to 1744 of SEQ ID NO: 169. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Penicillium emersonii NN051602.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 171 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving cellobiohydrolase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellobiohydrolase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 171. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Penicilliumpinophilum NN046877. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 171. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 58 to 1701 of SEQ ID NO: 171. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Penicillium pinophilum NN046877.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase I activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 19 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase I activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase I activity comprises or consists of the nucleotidesequence of SEQ ID NO: 19. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus terreusATCC 28865. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 19. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 64 to 1502 of SEQ ID NO: 19. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus terreus ATCC 28865.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 21 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 21. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Trichoderma reeseiRutC30. In another aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 21. In anotheraspect, the nucleotide sequence comprises or consists of nucleotides 64to 1254 of SEQ ID NO: 21. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in Trichoderma reesei RutC30.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 23 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 23. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pCIC161 whichis contained in E. coli NRRL B-30902. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 23. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 67 to 1185 of SEQ ID NO: 23. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pCIC161 which iscontained in E. coli NRRL B-30902.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 25 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingendoglucanase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 25. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Thermoascusaurantiacus CGMCC 0670. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 25. In another aspect, the nucleotide sequence comprises orconsists of nucleotides 91 to 1005 of SEQ ID NO: 25. In another aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Thermoascus aurantiacus CGMCC 0670.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 173 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving endoglucanase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 173. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in p Aspergillusfumigatus NN051616. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence of SEQ ID NO: 173.In another aspect, the nucleotide sequence comprises or consists ofnucleotides 55 to 1230 of SEQ ID NO: 173. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus fumigatus NN051616.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 175 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving endoglucanase II activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingendoglucanase II activity comprises or consists of the nucleotidesequence of SEQ ID NO: 175. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Neosartorya fischeriNRRL 181. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 175. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 49 to 1378 of SEQ ID NO: 175. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Neosartorya fischeri NRRL 181.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 27 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingbeta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 27. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pEJG113 whichis contained in E. coli NRRL B-30695. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 27. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 58 to 2580 of SEQ ID NO: 27. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pEJG113 which iscontained in E. coli NRRL B-30695.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 29 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingbeta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 29. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pKKAB whichis contained in E. coli NRRL B-30860. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 29. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 171 to 2753 of SEQ ID NO: 29. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pKKAB which iscontained in E. coli NRRL B-30860.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 31 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingbeta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 31. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus niger IBT10140. In another aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 31. In anotheraspect, the nucleotide sequence comprises or consists of nucleotides 58to 2934 of SEQ ID NO: 31. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in Aspergillus niger IBT 10140.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 177 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 177. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus aculeatusWDCM190. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 177. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 58 to 2937 of SEQ ID NO: 177. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus aculeatus WDCM190.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 179 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 179. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus kawashiiIFO4308. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 179. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 58 to 2932 of SEQ ID NO: 179. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus kawashii IFO4308.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 181 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 181. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in p Aspergillusclavatus NRRL 1. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 181. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 55 to 3059 of SEQ ID NO: 181. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus clavatus NRRL 1.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 183 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 183. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Thielavia terrestrisNRRL 8126. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 183. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 55 to 3029 of SEQ ID NO: 183. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Thielavia terrestris NRRL 8126.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 185 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 185. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in pUC19 D55EX which iscontained in E. coli NRRL B-50395. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 185. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 64 to 2790 of SEQ ID NO: 185. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in pUC19 D55EX which is containedin E. coli NRRL B-50395.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 187 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 187. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Penicillium oxalicum.In another aspect, the nucleotide sequence comprises or consists of themature polypeptide coding sequence of SEQ ID NO: 187. In another aspect,the nucleotide sequence comprises or consists of nucleotides 64 to 2790of SEQ ID NO: 187. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence contained inPenicillium oxalicum.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 189 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-glucosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-glucosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 189. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Talaromyces emersoniiCBS 549.92. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 189. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 58 to 2961 of SEQ ID NO: 189. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Talaromyces emersonii CBS 549.92.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 33 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 33. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pDZA2-7 whichis contained in E. coli NRRL B-30704. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 33. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 67 to 796 of SEQ ID NO: 33. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pDZA2-7 which iscontained in E. coli NRRL B-30704.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 35 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 35. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pTter61Ewhich is contained in E. coli NRRL B-30814. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 35. In another aspect, the nucleotidesequence comprises or consists of nucleotides 58 to 900 of SEQ ID NO:35. In another aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTter61Ewhich is contained in E. coli NRRL B-30814.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 37 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 37. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus fumigatusNN051616. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 37. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 64 to 859 of SEQ ID NO: 37. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus fumigatus NN051616.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 39 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 39. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 39. In another aspect, the nucleotidesequence comprises or consists of nucleotides 64 to 1018 of SEQ ID NO:39. In another aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 41 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 41. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence of SEQ ID NO: 41. In another aspect, thenucleotide sequence comprises or consists of nucleotides 76 to 832 ofSEQ ID NO: 41. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 43 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingcellulolytic enhancing activity comprises or consists of the nucleotidesequence of SEQ ID NO: 43. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pAG68 whichis contained in E. coli NRRL B-50320. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 43. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 64 to 1104 of SEQ ID NO: 43. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pAG68 which iscontained in E. coli NRRL B-50320.

In another fourth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity comprise or consistof nucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 191 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a GH61polypeptide having cellulolytic enhancing activity.

In one aspect, the isolated polynucleotide encoding a GH61 polypeptidehaving cellulolytic enhancing activity comprises or consists of thenucleotide sequence of SEQ ID NO: 191. In another aspect, the nucleotidesequence comprises or consists of the sequence contained inpGEM-T-GH61a51486 which is contained in E. coli DSM 22656. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence of SEQ ID NO: 191. In another aspect, thenucleotide sequence comprises or consists of nucleotides 67 to 868 ofSEQ ID NO: 191. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence contained inpGEM-T-GH61a51486 which is contained in E. coli NRRL DSM 22656.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 45 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 45. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in Aspergilluε aculeatus CBS 101.43.In another aspect, the nucleotide sequence comprises or consists of themature polypeptide coding sequence of SEQ ID NO: 45. In another aspect,the nucleotide sequence comprises or consists of nucleotides 69 to 1314of SEQ ID NO: 45. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence contained inAspergilluε aculeatus CBS 101.43.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 47 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 47. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in plasmid pHyGe009 which iscontained in E. coli NRRL B-30703. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 47. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 107 to 1415 of SEQ ID NO: 47. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pHyGe009 which iscontained in E. coli NRRL B-30703.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 49 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 49. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in plasmid pTF12Xy1170 which iscontained in E. coli NRRL B-50309. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 49. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 58 to 1194 of SEQ ID NO: 49. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pTF12Xy1170 which iscontained in E. coli NRRL B-50309.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 51 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 51. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in plasmid pGEM-T-Ppin3 which iscontained in E. coli DSM 22922. In another aspect, the nucleotidesequence comprises or consists of the mature polypeptide coding sequenceof SEQ ID NO: 51. In another aspect, the nucleotide sequence comprisesor consists of nucleotides 58 to 1439 of SEQ ID NO: 51. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pGEM-T-Ppin3 which iscontained in E. coli DSM 22922.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 53 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 53. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in Thielavia terrestris NRRL 8126. Inanother aspect, the nucleotide sequence comprises or consists of themature polypeptide coding sequence of SEQ ID NO: 53. In another aspect,the nucleotide sequence comprises or consists of nucleotides 58 to 1185of SEQ ID NO: 53. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence contained inThielavia terrestris NRRL 8126.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 193 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 193. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in Talaromyces emersonii NN05002. Inanother aspect, the nucleotide sequence comprises or consists of themature polypeptide coding sequence of SEQ ID NO: 193. In another aspect,the nucleotide sequence comprises or consists of nucleotides 70 to 1383of SEQ ID NO: 193. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence contained inTalaromyces emersonii NN05002.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 195 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 195. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in pMMar26 which is contained in E.coli NRRL B-50266. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence of SEQ ID NO: 195.In another aspect, the nucleotide sequence comprises or consists ofnucleotides 70 to 1384 of SEQ ID NO: 195. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in pMMar26 which is contained in E. coli NRRLB-50266.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 197 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode a polypeptide havingxylanase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 197. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in E. coli DSM 10361. In anotheraspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence of SEQ ID NO: 197. In another aspect, thenucleotide sequence comprises or consists of nucleotides 58 to 1188 ofSEQ ID NO: 197. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence contained in E. coliDSM 10361.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 55 of preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 55. In another aspect, the nucleotide sequence comprises orconsists of the sequence contained in Thermobifida fusca DSM 22883. Inanother aspect, the nucleotide sequence comprises or consists of themature polypeptide coding sequence of SEQ ID NO: 55. In another aspect,the nucleotide sequence comprises or consists of nucleotides 127 to 1014of SEQ ID NO: 55. In another aspect, the nucleotide sequence comprisesor consists of the mature polypeptide coding sequence contained inThermobifida fusca DSM 22883.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity comprise or consist of nucleotidesequences that have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 199 or SEQ ID NO: 304 of preferably atleast 80%, more preferably at least 85%, even more preferably at least90%, most preferably at least 95%, and even most preferably at least96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having xylanase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingxylanase activity comprises or consists of the nucleotide sequence ofSEQ ID NO: 199 or SEQ ID NO: 304. In another aspect, the nucleotidesequence comprises or consists of the sequence contained in Dictyoglomusthermophilum ATCC 35947. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequence of SEQID NO: 199 or SEQ ID NO: 304. In another aspect, the nucleotide sequencecomprises or consists of nucleotides 76 to 1137 of SEQ ID NO: 199 ornucleotides 85 to 693 of SEQ ID NO: 304. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Dictyoglomus thermophilum ATCC 35947.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 57 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-xylosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 57. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Trichoderma reeseiRutC30. In another aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 57. In anotheraspect, the nucleotide sequence comprises or consists of nucleotides 61to 2391 of SEQ ID NO: 57. In another aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in Trichoderma reesei RutC30.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 59 of preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which encode an active polypeptide.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-xylosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 59. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Talaromyces emersoniiCBS 393.64. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 59. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 64 to 2388 of SEQ ID NO: 59. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Talaromyces emersonii CBS 393.64.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 201 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-xylosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-xylosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 201. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus aculeatusCBS 172.66. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 201. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 52 to 2409 of SEQ ID NO: 201. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus aculeatus CBS 172.66.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 203 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-xylosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-xylosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 203. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus aculeatusCBS 186.67. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 203. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 52 to 2451 of SEQ ID NO: 203. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus aculeatus CBS 186.67.

In another fourth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity comprise or consist ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 205 of preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving beta-xylosidase activity.

In one aspect, the isolated polynucleotide encoding a polypeptide havingbeta-xylosidase activity comprises or consists of the nucleotidesequence of SEQ ID NO: 205. In another aspect, the nucleotide sequencecomprises or consists of the sequence contained in Aspergillus fumigatusNN051616. In another aspect, the nucleotide sequence comprises orconsists of the mature polypeptide coding sequence of SEQ ID NO: 205. Inanother aspect, the nucleotide sequence comprises or consists ofnucleotides 61 to 2376 of SEQ ID NO: 205. In another aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in Aspergillus fumigatus NN051616.

In a fifth aspect, the isolated polynucleotides encoding polypeptideshaving cellobiohydrolase I activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomicDNA sequence of the mature polypeptide coding sequence of SEQ ID NO: 1,or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:3, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 3, or (iii) a full-length complementary strand of(i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:5, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 5, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:7, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 7, or (iii) a full-length complementary strand of(i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:157, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 157, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:159, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 159, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:161, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 161, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:163, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 163, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase I activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:165, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 165, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:9, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 9, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:11, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 11, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:13, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 13, or (iii) a full-length complementary strandof (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:15, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 15, or (iii) a full-length complementary strandof (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:17, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 17, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:167, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 167, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:169, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 169, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having cellobiohydrolase II activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:171, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 171, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase I activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 19, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 19, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 21, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 21, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 23, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 23, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 25, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 25, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 173, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 173, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having endoglucanase II activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 175, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 175, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 27, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 27, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 29, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 29, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 31, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 31, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 177, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 177, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 179, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 179, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 181, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 181, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 183, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 183, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 185, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 185, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 187, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 187, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-glucosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 189, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 189, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:33, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 33, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:35, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 35, or (iii) a full-length complementary strandof (i) or (ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:37, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 37, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:39, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 39, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:41, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 41, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:43, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 43, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encoding GH61polypeptides having cellulolytic enhancing activity hybridize underpreferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:191, (ii) the cDNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 191, or (iii) a full-length complementary strand of (i) or(ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 45, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 45, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 47, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 47, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 49, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 49, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 51, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 51, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 53, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 53, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 193, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 193, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 195, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 195, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 197, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 197, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having xylanase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 55 or its full-lengthcomplementary strand. In another fifth aspect, the isolatedpolynucleotides encoding polypeptides having xylanase activity hybridizeunder preferably medium-high stringency conditions, more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:199 or SEQ ID NO: 304, (ii) the cDNA sequence of the mature polypeptidecoding sequence of SEQ ID NO: 199 or SEQ ID NO: 304, or (iii) afull-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 57, (ii) thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 57, or (iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 59, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 59, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 201, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 201, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 203, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 203, or(iii) a full-length complementary strand of (i) or (ii).

In another fifth aspect, the isolated polynucleotides encodingpolypeptides having beta-xylosidase activity hybridize under preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 205, (ii) the cDNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 205, or(iii) a full-length complementary strand of (i) or (ii).

Sources of Polypeptides Having Cellobiohydrolase I, CellobiohydrolaseII, Endoglucanase I, Endoglucanase II, Beta-Glucosidase, CellulolyticEnhancing, Xylanase, or Beta-Xylosidase Activity

A polypeptide having cellobiohydrolase I, cellobiohydrolase II,endoglucanase I, endoglucanase II, beta-glucosidase, cellulolyticenhancing, xylanase, or beta-xylosidase activity may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide having cellobiohydrolase I, cellobiohydrolase II,endoglucanase I, endoglucanase II, beta-glucosidase, cellulolyticenhancing, xylanase, or beta-xylosidase activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide.

A polypeptide having cellobiohydrolase I, cellobiohydrolase II,endoglucanase I, endoglucanase II, beta-glucosidase, cellulolyticenhancing activity, xylanase, or beta-xylosidase may also be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Chaetomium thermophilum CGMCC 0581 Cel7A polypeptide havingcellobiohydrolase I activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 2.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Myceliophthora thermophila CBS 117.65 Cel7A polypeptide havingcellobiohydrolase I activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 4.

In another aspect, the polypeptide having cellobiohydrolase I activityis an Aspergillus fumigatus NN055679 Cel7A polypeptide havingcellobiohydrolase I activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 6.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Thermoascus aurantiacus CGMCC 0583 Cel7A polypeptide havingcellobiohydrolase I activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 8.

In another aspect, the polypeptide having cellobiohydrolase I activityis a Penicillium emersonii NN051602 Cel7 polypeptide havingcellobiohydrolase I activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 158.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis a Penicillium pinophilum NN046877 Cel7 polypeptide havingcellobiohydrolase 1 activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 160.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis an Aspergillus terreus ATCC 28865 Cel7 polypeptide havingcellobiohydrolase 1 activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 162.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis a Neosartorya fischeri NRRL 181 Cel7 polypeptide havingcellobiohydrolase 1 activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 164.

In another aspect, the polypeptide having cellobiohydrolase 1 activityis an Aspergillus nidulans FGSCA4 Cel7 polypeptide havingcellobiohydrolase 1 activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 166.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Myceliophthora thermophila CBS 117.65 Cel6A polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 10.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Myceliophthora thermophila CBS 202.75 Cel6B polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 12.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Thielavia terrestris NRRL 8126 Cel6A polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 14.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Trichophaea saccata CBS 804.70 Cel6 polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 16.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Aspergillus fumigatus NN055679 Cel6A polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 18.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Fennellia nivea NN046949 Cel6 polypeptide having cellobiohydrolaseII activity, i.e., the polypeptide comprising the mature polypeptide ofSEQ ID NO: 168.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Penicillium emersonii NN051602 Cel6A polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 170.

In another aspect, the polypeptide having cellobiohydrolase II activityis a Penicillium pinophilum NN046877 Cel6A polypeptide havingcellobiohydrolase II activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 172.

In another aspect, the polypeptide having endoglucanase I activity is anAspergillus terreus ATCC 28865 Cel6A polypeptide having endoglucanase Iactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 20.

In another aspect, the polypeptide having endoglucanase II activity is aTrichoderma reesei RutC30 Cel5A polypeptide having endoglucanase IIactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 22.

In another aspect, the polypeptide having endoglucanase II activity is aMyceliophthora thermophila CBS 202.75 Cel5A polypeptide havingendoglucanase II activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 24.

In another aspect, the polypeptide having endoglucanase II activity is aThermoascus aurantiacus CGMCC 0670 Cel5A polypeptide havingendoglucanase II activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 26.

In another aspect, the polypeptide having endoglucanase II activity isan Aspergillus fumigatus NN051616 Cel5 polypeptide having endoglucanaseII activity, i.e., the polypeptide comprising the mature polypeptide ofSEQ ID NO: 174.

In another aspect, the polypeptide having endoglucanase II activity is aNeosartorya fischeri NRRL 181 polypeptide having endoglucanase IIactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 176.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus fumigatus NN055679 Cel5A polypeptide havingbeta-glucosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 28.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium brasilianum IBT 20888 Cel5A polypeptide havingbeta-glucosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus niger IBT 10140 GH3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 32.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus aculeatus WDCM190 Cel3 polypeptide havingbeta-glucosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 178.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus kawashii IFO4308 Cel3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 180.

In another aspect, the polypeptide having beta-glucosidase activity isan Aspergillus clavatus NRRL 1 Cel3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 182.

In another aspect, the polypeptide having beta-glucosidase activity is aThielavia terrestris NRRL 8126 Cel3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 184.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium oxalicum IBT5387 Cel3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 186.

In another aspect, the polypeptide having beta-glucosidase activity is aPenicillium oxalicum IBT5387 Cel3 polypeptide having beta-glucosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 188.

In another aspect, the polypeptide having beta-glucosidase activity is aTalaromyces emersonii CBS 549.92 Cel3 polypeptide havingbeta-glucosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 190.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thermoascus aurantiacus CGMCC 0583 GH61A polypeptidehaving cellulolytic enhancing activity, i.e., the polypeptide comprisingthe mature polypeptide of SEQ ID NO: 34.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thielavia terrestris NRRL 8126 GH61E polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 36.

In another aspect, the polypeptide having cellulolytic enhancingactivity is an Aspergillus fumigatus NN051616 GH61B polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 38.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Penicillium pinophilum NN046877 GH61A polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 40.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Penicillium sp. NN051602 GH61A polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 42.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thielavia terrestris NRRL 8126 GH61N polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 44.

In another aspect, the polypeptide having cellulolytic enhancingactivity is a Thermoascus crustaceus CBS 181.67 GH61A polypeptide havingcellulolytic enhancing activity, i.e., the polypeptide comprising themature polypeptide of SEQ ID NO: 192.

In another aspect, the polypeptide having xylanase activity is anAspergillus aculeatus CBS 101.43 polypeptide having xylanase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:46.

In another aspect, the polypeptide having xylanase activity is anAspergillus fumigatus NN055679 polypeptide having xylanase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:48.

In another aspect, the polypeptide having xylanase activity is aTrichophaea saccata CBS 804.70 polypeptide having xylanase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:50.

In another aspect, the polypeptide having xylanase activity is aPenicillium pinophilum NN046877 polypeptide having xylanase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:52.

In another aspect, the polypeptide having xylanase activity is aThielavia terrestris NRRL 8126 polypeptide having xylanase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:54.

In another aspect, the polypeptide having xylanase activity is aTalaromyces emersonii NN050022 polypeptide having xylanase activity,i.e., the polypeptide comprising of the mature polypeptide of SEQ ID NO:194.

In another aspect, the polypeptide having xylanase activity is aPenicillium sp. NN51602 polypeptide having xylanase activity, i.e., thepolypeptide comprising of the mature polypeptide of SEQ ID NO: 196.

In another aspect, the polypeptide having xylanase activity is aMeripilus giganteus CBS 521.95 polypeptide having xylanase activity,i.e., the polypeptide comprising of the mature polypeptide of SEQ ID NO:198.

In another aspect, the polypeptide having xylanase activity is aThermobifida fusca DSM 22883 polypeptide having xylanase activity, i.e.,the polypeptide comprising the mature polypeptide of SEQ ID NO: 56.

In another aspect, the polypeptide having xylanase activity is aDictyoglomus thermophilum ATCC 35947 polypeptide having xylanaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 200 or SEQ ID NO: 305.

In another aspect, the polypeptide having beta-xylosidase activity is aTrichoderma reesei RutC30 polypeptide having beta-xylosidase activity,i.e., the polypeptide comprising the mature polypeptide of SEQ ID NO:58.

In another aspect, the polypeptide having beta-xylosidase activity is aTalaromyces emersonii CBS 393.64 polypeptide having beta-xylosidaseactivity, i.e., the polypeptide comprising the mature polypeptide of SEQID NO: 60.

In another aspect, the Family 3 polypeptide having beta-xylosidaseactivity is an Aspergillus aculeatus CBS 172.66 polypeptide havingbeta-xylosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 202.

In another aspect, the Family 3 polypeptide having beta-xylosidaseactivity is an Aspergillus aculeatus CBS 186.67 polypeptide havingbeta-xylosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 204.

In another aspect, the Family 3 polypeptide having beta-xylosidaseactivity is an Aspergillus fumigatus NN051616 polypeptide havingbeta-xylosidase activity, i.e., the polypeptide comprising the maturepolypeptide of SEQ ID NO: 206.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotideencoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Such polypeptides also include fused polypeptides or cleavable fusionpolypeptides in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide or fragment thereof. A fusedpolypeptide is produced by fusing a nucleotide sequence (or a portionthereof) encoding another polypeptide to a nucleotide sequence (or aportion thereof) of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andthat expression of the fused polypeptide is under control of the samepromoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Nucleic Acid Constructs

A nucleic acid construct comprising an isolated polynucleotide encodinga polypeptide component of an enzyme composition of the presentinvention may be constructed by operably linking the polynucleotide toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any nucleotide sequence that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus nidulans acetamidase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase, Fusariumoxysporum trypsin-like protease (WO 96/00787), Fusarium venenatumamyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900),Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase,Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters from the gene encoding neutral alpha-amylase in Aspergillusniger in which the untranslated leader has been replaced by anuntranslated leader from the gene encoding triose phosphate isomerase inAspergillus nidulans or Aspergillus oryzae); and mutant, truncated, andhybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The various nucleic acids and control sequences described herein may bejoined together to construct a recombinant expression vector that mayinclude one or more (several) convenient restriction sites to allow forinsertion or substitution of an isolated polynucleotide encoding apolypeptide component of the enzyme composition at such sites.Alternatively, a polynucleotide sequence may be expressed by insertingthe nucleotide sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors preferably contain one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of the gene product. An increase in the copynumber of the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingone or more isolated polynucleotides encoding polypeptide components ofthe enzyme composition, which are advantageously used in the recombinantproduction of the polypeptides. A vector is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M.I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing an enzymecomposition of the present invention, comprising: (a) cultivating arecombinant host cell, as described herein, under conditions conducivefor production of the enzyme composition; and (b) recovering the enzymecomposition.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of the enzymecomposition using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymecomposition to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the components of the enzyme composition are secretedinto the nutrient medium, the enzyme composition can be recovereddirectly from the medium. If the components of the enzyme compositionare not secreted into the medium, the enzyme composition can berecovered from cell lysates.

The polypeptide components may be detected using methods known in theart that are specific for the polypeptides. These detection methods mayinclude use of specific antibodies, formation of an enzyme product, ordisappearance of an enzyme substrate.

The resulting enzyme composition may be recovered using methods known inthe art. For example, the enzyme composition may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray-drying, evaporation,or precipitation.

An enzyme composition of the present invention may be purified by avariety of procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Methods of Processing Cellulosic Material

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition of the present invention. In apreferred aspect, the method further comprises recovering the degradedor converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition of the present invention; (b)fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition of the present invention. In apreferred aspect, the fermenting of the cellulosic material produces afermentation product. In another preferred aspect, the method furthercomprises recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify acellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., chemicals and fuels. Theproduction of a desired fermentation product from cellulosic materialtypically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF), (hybrid hydrolysis and fermentation (HHCF), anddirect microbial conversion (DMC). SHF uses separate process steps tofirst enzymatically hydrolyze lignocellulose to fermentable sugars,e.g., glucose, cellobiose, cellotriose, and pentose sugars, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of lignocellulose and the fermentation of sugars to ethanolare combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, lignocellulose hydrolysis, and fermentation) in oneor more steps where the same organism is used to produce the enzymes forconversion of the lignocellulose to fermentable sugars and to convertthe fermentable sugars into a final product (Lynd, L. R., Weimer, P. J.,van Zyl, W. H., and Pretorius, I. S., 2002, Microbial celluloseutilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.Reviews 66: 506-577). It is understood herein that any method known inthe art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt the plantcell wall components (Chandra et al., 2007, Substrate pretreatment: Thekey to effective enzymatic hydrolysis of lignocellulosics? Adv. Biochem.Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment oflignocellulosic materials for efficient bioethanol production, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Pretreatments to enhance the digestibility of lignocellulosic biomass,Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features ofpromising technologies for pretreatment of lignocellulosic biomass,Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008,Pretreatment of lignocellulosic wastes to improve ethanol and biogasproduction: A review, Int. J. of Mol. Sci. 9: 1621-1651; Yang and Wyman,2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol,Biofuels Bioproducts and Biorefining-Biofpr 2: 26-40).

Mechanical Pretreatment. The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling) to disrupt and/or reduce particle sizeplant cellwall components of the cellulosic material.

Chemical Pretreatment. In practicing the methods of the presentinvention, any chemical pretreatment process known in the art can beused to disrupt plant cell wall components of the cellulosic material(Chandra et al., 2007, supra; Galbe and Zacchi, 2007, Pretreatment oflignocellulosic materials for efficient bioethanol production, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Pretreatments to enhance the digestibility of lignocellulosic biomass,Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features ofpromising technologies for pretreatment of lignocellulosic biomass,Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008,Pretreatment of lignocellulosic wastes to improve ethanol and biogasproduction: A review, Int. J. of Mol. Sci. 9: 1621-1651; Yang and Wyman,2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol,Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).

Conventional chemical pretreatments include, but are not limited to,steam pretreatment (with or without explosion), dilute acidpretreatment, hot water pretreatment, alkaline pretreatment, limepretreatment, wet oxidation, wet explosion, ammonia fiber explosion,organosolv pretreatment, and biological pretreatment. Additionalpretreatments include ammonia percolation, ultrasound, electroporation,microwave, supercritical CO₂, supercritical H₂O, ozone, and gammairradiation pretreatments.

The cellulosic material can be chemically pretreated before hydrolysisand/or fermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, cellobiose, and/or xylose. In most cases thepretreatment step itself can result in some conversion of the cellulosicmaterial to fermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulosic material is mixed withdilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulosic material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Physical Pretreatment. The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from the cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, physical pretreatmentis performed in a batch-process, steam gun hydrolyzer system that useshigh pressure and high temperature as defined above, e.g., a SundsHydrolyzer available from Sunds Defibrator AB, Sweden.

The cellulosic material can be subjected to pre-soaking, wetting,washing, or conditioning prior to pretreatment using methods known inthe art.

Combined Physical and Chemical Pretreatment: The cellulosic material canbe pretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and alternatively also hemicelluloseto fermentable sugars, such as glucose, cellobiose, xylose, xylulose,arabinose, mannose, galactose, and/or soluble oligosaccharides. Thehydrolysis is performed enzymatically by an enzyme composition of thepresent invention.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

In a preferred aspect, an effective amount of an enzyme composition ofthe present invention is about 0.5 to about 50 mg, preferably at about0.5 to about 40 mg, more preferably at about 0.5 to about 30 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 1.0 to about 10 mg, andmost preferably at about 2.0 to about 5 mg per g of cellulose in acellulosic material.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); aketone (e.g., acetone); an amino acid (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and a gas (e.g., methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

Minimal medium plates were composed of 6 g of NaNO₃, 0.52 g of KCl, 1.52g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Noble agar,20 ml of 50% glucose, 2.5 ml of MgSO₄.7H₂O, 20 ml of a 0.02% biotinsolution, and deionized water to 1 liter.

COVE trace elements solution was composed of 0.04 g of Na₂B₄O₇.10H₂O,0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, and 0.5 ml of AMG trace metals solution; pH 5.0.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.7H₂O, and 3 g of citric acid.

NNCYP-PCS medium was composed of 5.0 g of NaNO₃, 3.0 g of NH₄Cl, 2.0 gof MES, 2.5 g of citric acid, 0.2 g of CaCl₂ 2H₂O, 1.0 g of BactoPeptone, 5.0 g of yeast extract, 0.2 g of MgSO₄ 7 H₂O, 4.0 g of K₂HPO₄,1.0 ml of COVE trace elements solution, 2.5 g of glucose, 25.0 g ofpretreated corn stover (PCS), and deionized water to 1 liter.

2×YT medium was composed per liter of 16 g of tryptone, 10 g of yeastextract, and 5 g of NaCl.

2×YT plates were composed per liter of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl and 15 g of Noble agar.

YG agar plates were composed per liter of 5.0 g of yeast extract, 10.0 gof glucose, and 20.0 g of agar.

YEG medium was composed per liter of 20 g of dextrose and 5 g of yeastextract.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of sodium chloride.

LB agar plates were composed of 10 g of tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of agar, and 1 liter of distilled water.

YP medium was composed per liter of 10 g of yeast extract and 20 g ofBacto peptone.

MEX-1 medium was composed per liter of 20 g of soya bean meal, 15 g ofwheat bran, 10 g of microcrystalline cellulose (AVICEL®; FMC,Philadelphia, Pa., USA), 5 g of maltodextrin, 3 g of Bactopeptone, 0.2 gof pluronic, and 1 g of olive oil.

LB ampicillin medium was composed per liter of 10 g of tryptone, 5 g ofyeast extract, 5 g of sodium chloride, and 50 mg of ampicillin (filtersterilized, added after autoclaving).

LB ampicillin plates were composed of 15 g of bacto agar per liter of LBampicillin medium.

MY25 medium was composed per liter of 25 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g ofurea, 10 g of yeast extract, and 1.5 ml of AMG trace metals solution,adjusted to pH 6.

YPD medium was composed of 1% yeast extract, 2% peptone, andfilter-sterilized 2% glucose added after autoclaving.

YPM medium was composed of 1% yeast extract, 2% peptone, andfilter-sterilized 2% maltodextrin added after autoclaving.

SC-URA medium with glucose or galactose was composed of 100 ml of 10×Basal salts, 25 ml of 20% casamino acids without vitamins, 10 ml of 1%tryptophan, 4 ml of 5% threonine (filter sterilized, added afterautoclaving), and 100 ml of 20% glucose or 100 ml of 20% galactose(filter sterilized, added after autoclaving), and deionized water to 1liter.

10× Basal salts solution was composed of 75 g of yeast nitrogen base,113 g of succinic acid, 68 g of NaOH, and deionized water to 1 liter.

SC-agar plates were composed of 20 g of agar per liter of SC-URA medium(with glucose or galactose as indicated).

0.1% AZCL xylan SC-URA agar plates with galactose were composed of 20 gof agar per liter of SC-URA medium with galactose and 0.1% AZCL oatxylan (Megazyme, Wicklow, Ireland).

SC-URA medium with galactose was composed of 900 ml of SC-Grund Agar(autoclaved), 4 ml of 5% threonine (filter sterilized), and 100 ml of20% galactose (filter sterilized).

SC-Grund Agar was composed of 7.5 g Yeast Nitrogen Base (without aminoacids), 11.3 g of succinic acid, 6.8 g of sodium hydroxide, 5.6 g ofcasamino acids, 0.1 g of L-tryptophan, 20 g of agar, and deionized waterto 1 liter.

COVE plates were composed per liter of 342.3 g of sucrose, 25 g of Nobleagar, 20 ml of COVE salts solution, 10 mM acetamide, and 15 or 20 mMCsCl. The solution was adjusted to pH 7.0 before autoclaving.

COVE2 plates were composed per liter of 30 g of sucrose, 20 ml of COVEsalts solution, 20 ml of 1 M acetamide, and 25 g of Agar Noble.

COVE salts solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂ PO₄, and 50 ml of COVE trace metals.

YPG medium was composed per liter of 10 g of yeast extract, 10 g ofBacto peptone, and 20 g of glucose.

M410 medium was composed per liter of 50 g of maltose, 50 g of glucose,2 g of MgSO₄-7H₂O, 2 g of KH₂PO₄, 4 g of citric acid anhydrous powder, 8g of yeast extract, 2 g of urea, 0.5 g of AMG trace metals solution, and0.5 g of CaCl₂ at pH 6.0.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄; sterilized by autoclaving andthen filter-sterilized glucose was added to 20 mM.

SY50 medium was composed per liter of 50 g of sucrose, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, anhydrous, 2 g of K₂SO₄, 2 g of citric acid,10 g of yeast extract, 2 g of urea, 0.5 g of CaCl₂.2H₂O, and 0.5 g of200×AMG trace metals solution, pH 6.0.

200×AMG trace metals solution was composed per liter of 3 g of citricacid, 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 13.8 g of FeSO₄.7H₂O,and 8.5 g of MnSO₄.H₂O.

Cal-18 medium was composed per liter of 40 g of yeast extract, 1.3 g ofmagnesium sulfate, 50 g of maltodextrin, 20 g of NaH₂PO₄, and 0.1 g ofantifoam.

Cellulase-inducing medium was composed of 20 g of cellulose, 10 g ofcorn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g ofCaCl₂, 0.42 g of MgSO₄.7H₂O, 0.42 ml of Trichoderma trace metalssolution, and 1-2 drops of antifoam.

Trichoderma trace metals solution was composed per liter of 216 g ofFeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O,2.4 g of H₃BO₃, and 336 g of citric acid.

TE was composed of 10 mM Tris pH 7.4 and 0.1 mM EDTA.

YPM medium contained 1% yeast extract, 2% of peptone, and 2% of maltosein deionized water.

MY50 medium was composed of 50 g of Maltodextrin, 2 g of MgSO47H₂O, 10 gof KH₂PO₄, 2 g of K2SO4, 2 g of citric acid, 10 g of yeast extract, 2 gof urea, 0.5 ml of AMG trace metals solution, and distilled water to 1liter.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO4.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.7H₂O, 3 g of citric acid, and distilled waterto 1 liter.

50× Vogels medium was composed per liter of 150 g of sodium citrate, 250g of KH₂PO₄, 10 g of MgSO₄.7H₂O, 10 g of CaCl₂.2H₂O, 2.5 ml of biotinstock solution, 5.0 ml of AMG trace metals solution, and distilled waterto 1 liter.

COVE agar selective plates were composed of 218 g sorbitol, 20 g agar,20 ml COVE salts solution, 10 mM acetamide, 15 mM CsCl, and deionizedwater to 1 liter. The solution was adjusted to pH 7.0 beforeautoclaving.

COVE salts solution was composed of 26 g KCl, 26 g MgSO₄.7H₂O, 76 gKH₂PO₄, 50 ml COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g Na₂B₄O₇.10H₂O, 0.4 gCuSO₄.5H₂O, 1.2 g FeSO₄.7H₂O, 0.7 g MnSO₄.H₂O, 0.8 g Na₂MoO₂.2H₂O, 10 gZnSO₄.7H₂O, and deionized water to 1 liter.

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone and 2%glucose in deionized water.

YP+2% maltodextrin medium is composed of 2% peptone, 2% maltodextrin,and 1% yeast extract in deionized water.

DAP-2C-1 medium is composed of 3% maltodextrin, 1.1% magnesium sulfate,0.52% tri-potassium phosphate, 0.2% citric acid, 0.1% potassiumdihydrogen phosphate, 0.1% Dowfax 63N10, 0.05% yeast extract, and 0.05%of a trace element solution (1.39% ferrous sulfate, 0.845% maganesesulfate, 0.68% zinc chloride, 0.3% citric acid, 0.25% copper sulfate,and 0.013% nickel chloride) in deionized water.

DAP-2C-1 medium is composed of 2% glucose, 1.1% magnesium sulfate, 1.0%maltose, 0.52% tri-potassium phosphate, 0.2% citric acid, 0.1% potassiumdihydrogen phosphate, 0.1% Dowfax 63N10, 0.05% yeast extract, and 0.05%of a trace element solution (1.39% ferrous sulfate, 0.845% maganesesulfate, 0.68% zinc chloride, 0.3% citric acid, 0.25% copper sulfate,and 0.013% nickel chloride) in deionized water.

Example 1: Preparation of Chaetomium thermophilum CGMCC 0581 Cel7ACellobiohydrolase I

The Chaetomium thermophilum CGMCC 0581 Cel7A cellobiohydrolase I (CBHI)gene (SEQ ID NO: 1 [DNA sequence] and SEQ ID NO: 2 [deduced amino acidsequence]) was isolated according to WO 2003/000941 and expressed inAspergillus oryzae JaL250 (WO 99/61651).

The fungal strain Chaetomium thermophilum CGMCC 0581 was grown on agarplate composed of 0.5% yeast extract, 1% glucose and 2% agar for 3 daysat 45° C. The fully grown culture was used to inoculate shake flaskscontaining liquid medium composed of 3% soymeal, 1.5% maltose, and 0.5%peptone. The flasks were incubated at 45° C. for 48 hours with shaking.The mycelia were harvested by centrifugation of the culture broth at8000 rpm, 4° C. for 30 minutes, transferred into a clean plastic bagfollowed by immediate freezing in liquid nitrogen, and stored at −80° C.before total RNA was isolated.

The frozen mycelia were grounded into a very fine powder with asterilized mortar and pestle baked at 200° C. for 24 hours. An RNEASY®Plant Mini Kit (QIAGEN Inc., Valencia, Calif., USA) was used to isolatetotal RNA according to the manufacturer's instructions.

First strand cDNA synthesis from the total RNA was performed using a 3′RACE System for Rapid Amplification of cDNA Ends (InvitrogenCorporation, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The first strand cDNA of 3′ RACE was used as PCR templatefor PCR screening.

Two oligonucleotides shown below were used for PCR screening of cDNA ofChaetomium thermophilum CGMCC 0581. The forward primer was derived froman alignment of conserved regions of cellobiohydrolase I genes and thereverse primer was provided by the 3′ RACE System.

Forward primer: (SEQ ID NO: 67) 5′-GGnACnGGnTA(t/c)TG(t/c)GA-3′Reverse primer: (SEQ ID NO: 68) 5′-GGCCACGCGTCGACTAGTAC-3′

One hundred picomoles of the forward primer and 10 picomoles of thereverse primer were used in a PCR reaction composed of 2 μl of the firststrand cDNA of 3′ RACE, 5 μl of 10×Taq DNA polymerase buffer (PromegaCorporation, Madison, Wis., USA), 3 μl of 25 mM MgCl₂, 1 μl of 10 mMdNTP, and 2.5 units of Taq DNA polymerase (Promega Corporation, Madison,Wis., USA) in a final volume of 50 μl. The amplification was performedin a thermocycler programmed for 1 cycle at 95° C. for 3 minutes; 30cycles each at 95° C. for 30 seconds, 50° C. for 30 seconds, and 72° C.for 50 seconds; and 1 cycle at 72C for 10 minutes. The heat block thenwent to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.3 kb product band was excisedfrom the gel, and purified using a WIZARD® PCR Preps DNA PurificationSystem (Promega Corporation, Madison, Wis., USA) according to themanufacturer's instructions. The PCR product was sequenced using a 377DNA Analyzer (Applied Biosystems Inc, Foster City, Calif., USA).Sequencing showed that the 1.3 kb fragment was homologous tocellobiohydrolase 1.

Two oligos shown below were designed for the 5′ end cloning of theChaetomium thermophilum CGMCC 0581 Cel7A cellobiohydrolase 1 by using a5′ RACE System for Rapid Amplification of cDNA Ends (InvitrogenCorporation, Carlsbad, Calif., USA).

Primer 4310AS1: (SEQ ID NO: 69) 5′-AGATATCCATCTCAGAGCA-3′Primer 4310AS2: (SEQ ID NO: 70) 5′-GTTGGCATCATTGGTCG-3′

The gene specific primer 4310AS1 was used for the first strand cDNAsynthesis using a 5′ RACE System according to the manufacturer'sinstructions. The first strand cDNA of 5′ RACE (5 μl) was used astemplate for PCR amplification composed of 5 μl of 10×Taq DNA polymerasebuffer, 3 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTP, 1 μl of 10 μM 4310AS2primer, 1 μl of 10 μM primer AAP (Abridged Anchor Primer, provided bythe kit), and 2.5 units of Taq DNA polymerase in a final volume of 50μl. The amplification was performed in a thermocycler programmed for 1cycle at 94° C. for 3 minutes; 30 cycles each at 95° C. for 30 seconds,50° C. for 30 seconds, and 72° C. for 50 seconds; and 1 cycle at 72° C.for 10 minutes. The heat block then went to a 4° C. soak cycle.

The PCR products were isolated by 1.0% agarose gel electrophoresis usingTBE buffer and purified using a WIZARD® PCR Preps DNA PurificationSystem. A dominant DNA fragment of 0.8 kb was confirmed to be the 5′ endof Chaetomium thermophilum CGMCC 0581 Cel7A cellobiohydrolase I gene bysequencing using a 377 DNA Analyzer.

Two primers shown below were designed based on the sequence informationfrom both 5′ and 3′ end cloning. They were used for full-length cloningof the Chaetomium thermophilum CGMCC 0581 Cel7A cellobiohydrolase Igene.

Primer 4310S: (SEQ ID NO: 71) 5′-ATCCTCTCCTTCCAGTTTTC-3′ Primer 4310AS:(SEQ ID NO: 72) 5′-TATCCAAGTAGTCCACAACC-3′

Ten picomoles of the above two primers were used in a PCR reactioncomposed of 5 μl of first strand cDNA of 3′ RACE, 5 μl of 10×Taq DNApolymerase buffer, 3 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTP, and 2.5units of Taq DNA polymerase in a final volume of 50 μl. Theamplification was performed in a thermocycler programmed for 1 cycle at95° C. for 3 minutes; 30 cycles each at 95° C. for 50 seconds, 55° C.for 50 seconds, and 72° C. for 90 seconds; and 1 cycle at 72° C. for 10minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.5 kb product band was excisedfrom the gel, and purified using a WIZARD® PCR Preps DNA PurificationSystem. The PCR fragment was then ligated to pGEM-T using a pGEM-TVector System (Promega Corporation, Madison, Wis., USA). The plasmid DNAwas confirmed by sequencing using a 377 DNA Analyzer. The correct clonewas designated pT43-10.

Two synthetic oligonucleotide primers containing Bsp HI sites at theirends, shown below, were designed to PCR amplify the full-length openreading frame of the Chaetomium thermophilum CGMCC 0581 Family GH7Acellobiohydrolase I gene. A Rapid Ligation Kit (Roche Applied Science,Indianapolis, Ind., USA) was used to clone the fragment into pAILo2 (WO2004/099228).

PCR Forward primer: (SEQ ID NO: 73) 5′-TCATGA TGTACAAGAAGTTCGCCG-3′PCR Reverse primer: (SEQ ID NO: 74) 5′-TCATGA TTACAGGCACTGGCTGTAC-3′Bold letters represent coding sequence. The underlined sequence containssequence identity to the BspHI restriction site.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 50 ng of plasmid pT43-10 containing the Chaetomiumthermophilum CGMCC 0581 cellobiohydrolase I gene, 1×Pwo AmplificationBuffer with MgSO₄ (Boehringer Mannheim, Indianapolis, Ind., USA), 4 μlof 10 mM blend of dATP, dTTP, dGTP, and dCTP, and 2.5 units of Pwo DNAPolymerase (Boehringer Mannheim, Indianapolis, Ind., USA), in a finalvolume of 50 μl. A DNA ENGINE™ Thermal Cycler (MJ Research, Waltham,Mass., USA) was used to amplify the fragment programmed for one cycle at94° C. for 2 minutes; 35 cycles each at 94° C. for 30 seconds, 62° C.for 30 seconds, and 72° C. for 1.5 minutes. After the 35 cycles, thereaction was incubated at 72° C. for 10 minutes and then cooled at 10°C. until further processed.

A 1.6 kb PCR reaction product was isolated on a 0.8% GTG® agarose gel(Cambrex Bioproducts, East Rutherford, N.J., USA) using 40 mM Trisbase-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer and 0.1 μg ofethidium bromide per ml. The DNA band was visualized with the aid of aDARKREADER™ Transilluminator (Clare Chemical Research, Dolores, Colo.,USA) to avoid UV-induced mutations. The 1.6 kb DNA band was excised witha disposable razor blade and purified with an ULTRAFREE® DA spin cup(Millipore, Billerica, Mass., USA) according to the manufacturer'sinstructions.

The purified PCR fragment was cloned into pCR®4Blunt-TOPO® (Invitrogen,Carlsbad, Calif., USA) using a TOPO® Blunt Cloning Kit (Invitrogen,Carlsbad, Calif., USA) according to the manufacturer's instructions. PCRclones containing the coding regions of interest were sequenced to PhredQ values of at least 40 to insure that there were no PCR induced errors.All sequence aligments were performed with Consed (University ofWashington). One of the clones was determined to have the expectedsequence and was selected and re-named CtPCR. The CtPCR clone containingthe C. thermophilum cellobiohydrolase I coding region was digested withBsp HI and gel purified as described above. This DNA fragment was thenligated into the Nco I restriction site of pAILo2 with a Rapid LigationKit. Expression clones were confirmed by restriction digestion andsequenced to confirm that the junction vector-insert was correct.Plasmid DNA for transformation was prepared with a Midi-Prep Kit (QIAGENInc., Valencia, Calif., USA). The final clone was re-named pAILo4.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Eightmicrograms of pAILo4 (as well as pAILo2 as a vector control) were usedto transform Aspergillus oryzae JaL250 protoplasts. Twelve transformantswere isolated to individual PDA plates and incubated for 5 days at 34°C. Confluent spore plates were washed with 5 ml of 0.01% TWEEN® 80 andthe spore suspension was used to inoculate 25 ml of MDU2BP medium in 125ml glass shake flasks. Transformant cultures were incubated at 34° C.with constant shaking at 200 rpm. At day five post-inoculation, cultureswere centrifuged at 6000×g and their supernatants collected. Fivemicroliters of each supernatant were mixed with an equal volume of 2×loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm8%-16% Tris-glycine SDS-PAGE gel and stained with SIMPLY BLUE™ SafeStain(Invitrogen, Carlsbad, Calif., USA). SDS-PAGE profiles of the culturebroths showed that twelve out of twelve transformants had a new proteinband of approximately 66 kDa. Transformant number 12 was selected anddesignated A. oryzae JaI250AILo4.

Shake flask medium was composed per liter of 50 g of glucose, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of K₂SO₄, 0.5 g of CaCl₂.2H₂O, 2 g ofcitric acid, 10 g of yeast extract, 0.5 g of AMG trace metals solution,and 2 g of urea. AMG trace metals solution was composed per liter of13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O and 3.0 g of citric acid monohydrate.

One hundred ml of shake flask medium was added to a 500 ml shake flask.The shake flask was inoculated with a glycerol spore stock of A. oryzaeJaI250AILo4 and incubated at 34° C. on an orbital shaker at 200 rpm for24 hours. Fifty ml of the shake flask broth was used to inoculate afermentation vessel.

Fermentation batch medium was composed per liter of 25 g of sucrose, 2 gof MgSO₄.7H₂O, 2 g of KH₂PO₄, 3 g of K₂SO₄, 5 g of (NH₄)₂HPO₄, 1 g ofcitric acid, 10 g of yeast extract, 0.5 g of AMG trace metals solution,and 0.55 g of pluronic antifoam. Fermentation feed medium was composedper liter of 320 g of maltose, 5 g of pluronic antifoam, and 1 g ofcitric acid monohydrate.

A total of 2 liters of the fermentation batch medium was added to aglass jacketed fermentor. The fermentation vessel was maintained at atemperature of 34° C. and the pH was controlled at 7 for 180 hours using10% NH₄OH and 10% H₃PO₄. Air was added to the vessel at a rate of 1 vvmand the broth was agitated by Rushton impeller rotating at 1100 rpm.Feed was started at a rate of 4 g per hour when the batch sucrose wasconsumed as indicated by a rise in the dissolved oxygen reading (atapproximately 18-24 hours). At the end of the fermentation, whole brothwas harvested from the vessel and centrifuged at 3000×g to remove thebiomass. The supernatant was sterile filtered and stored at 5 to 10° C.

A 350 ml (3.15 g total protein) aliquot of the filtered A. oryzaeJaI250AILo4 fermentation broth (AOC18-7) containing recombinantChaetomium thermophilum Cel7A cellobiohydrolase I was concentrated anddesalted, and then purified over a Q SEPHAROSE® Big Bead column (GEHealthcare, Piscataway, N.J., USA) in 20 mM Tris-HCl pH 8, over a linear0 to 1 M NaCl gradient. Fractions were pooled based on SDS-PAGE,concentrated and buffer-exchanged to 25 mM Tris-HCl, pH 8. The purifiedcellobiohydrolase I (approximately 800 mg total) was approximately 90%pure by SDS-PAGE. Protein concentration was determined using aMicroplate BOA™ Protein Assay Kit (Thermo Fischer Scientific, Waltham,Mass., USA) in which bovine serum albumin was used as a proteinstandard.

Example 2: Preparation of Myceliophthora thermophila CBS 117.65 Cel7ACellobiohydrolase I

The Myceliophthora thermophila CBS 117.65 Cel7A cellobiohydrolase I(CBHI) gene (SEQ ID NO: 3 [DNA sequence] and SEQ ID NO: 4 [deduced aminoacid sequence]) was isolated according to WO 2003/000941 and expressedin Aspergillus oryzae JaL250.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the full-length open reading frame from Myceliophthorathermophila CBS 117.65 encoding the Family Cel7A cellobiohydrolase I.

PCR Forward primer: (SEQ ID NO: 75) 5′-ctcgcagtcgcagtcaag-3′ PCR Reverse primer: (SEQ ID NO: 76) 5′-cggtcaggttgcagtttag-3′ 

Thirty picomoles of each of the primers above were used in anamplification reaction containing 50 ng of DNA consisting of a pool ofMyceliophtora thermophila CBS 117.65 cDNA prepared according to U.S.Pat. No. 6,242,237, 1× EXPAND™ PCR Buffer (Roche Diagnostics, Mannheim,Germany), 4 μl of 2.5 mM blend of dATP, dTTP, dGTP, and dCTP, 0.75 μl ofEXPAND™ DNA Polymerase (Roche Diagnostics, Mannheim, Germany), in afinal volume of 50 μl. The amplification of the fragment was performedin a thermocycler programmed for one cycle at 94° C. for 5 minutes; and35 cycles each at 94° C. for 1 minute, 54° C. for 1 minute, and 72° C.for 2 minutes. After the 30 cycles, the reaction was incubated at 72° C.for 10 minutes and then cooled at room temperature until furtherprocessed.

A 1.3 kb PCR product was isolated by 1% agarose gel electrophoresisusing TBE buffer and 0.1 μg of ethidium bromide per ml. The 1.3 kb DNAband was excised with a disposable razor blade and purified using aJETSORB Gel Extraction Kit (Genomed GmbH, Löhne, Germany) according tothe manufacturer's instructions.

The purified PCR fragment was cloned into pCR®4Blunt-TOPO® according tothe manufacturer's instructions. PCR clones containing the codingregions of interest were sequenced. One of the clones having theexpected sequence was selected and named pDAu27 #15.

Two synthetic oligonucleotide primers containing a Bsp HI restrictionsite on the forward primer and a Pac I restriction site on the reverseprimer, shown below, were designed to PCR amplify the full-length openreading frame of the Myceliophthora thermophila CBS 117.65 Cel7Acellobiohydrolase I from pDAu27 #15. A Rapid Ligation Kit was used toclone the fragment into pAILo2 (WO 2004/099228).

PCR Forward primer: (SEQ ID NO: 77) 5′-TCATGA AGCAGTACCTCCAGTA-3′PCR Reverse primer: (SEQ ID NO: 78) 5′-TTAATTAA TTAGACGTTGACAGTCGAGC-3′Bold letters represent coding sequence. The underlined sequence containssequence identity to the Bsp HI and Pac I restriction sites.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 50 ng of plasmid pDAu27 #15 containing the Myceliophthorathermophila CBS 117.65 cellobiohydrolase I I gene, 1×Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of 10 mM blend of dATP,dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA Polymerase(Invitrogen, Carlsbad, Calif., USA), 1 μl of 50 mM MgSO4, and 2.5 μl of10×pCRx Enhancer solution (Invitrogen, Carlsbad, Calif., USA) in a finalvolume of 50 μl. A DNA ENGINE™ Thermal Cycler was used to amplify thefragment programmed for one cycle at 98° C. for 2 minutes; and 35 cycleseach at 94° C. for 30 seconds, 58° C. for 30 seconds, and 68° C. for 1.5minutes. After the 35 cycles, the reaction was incubated at 68° C. for10 minutes and then cooled at 10° C. until further processed.

A 1.3 kb PCR reaction product was isolated on a 0.8% GTG® agarose gelusing TAE buffer and 0.1 μg of ethidium bromide per ml. The DNA band wasvisualized with the aid of a DARKREADER™ Transilluminator to avoidUV-induced mutations. The 1.3 kb DNA band was excised with a disposablerazor blade and purified with an ULTRAFREE® DA spin cup according to themanufacturer's instructions.

The purified PCR fragment was cloned into pCR®4Blunt-TOPO® according tothe manufacturer's instructions. PCR clones containing the coding regionof interest were sequenced to Phred Q values of at least 40 to insurethat there were no PCR induced errors. All sequence aligments wereperformed with Consed (University of Washington). One of the clones thatwas shown to have the expected sequence was selected and re-named MtPCR.The MtPCR clone containing the M. thermophila cellobiohydrolase I codingregion was double digested with Bsp HI and Bss SI and a 352 bp fragmentwas gel purified as described above. Another aliquot of MtPCR was alsodouble digested with Bss SI and Pac I and a 1009 bp fragment was gelpurified as described above. These DNA fragments were then ligated intopAILo2 previously digested with Nco I and Pac I in a three-way ligationusing a Rapid Ligation Kit. Expression clones were confirmed byrestriction digestion and sequenced to confirm that the junctionvector-insert was correct. Plasmid DNA for transformation was preparedwith a Midi-Prep Kit. The final clone was re-named pAILo10.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Six micrograms of pAILo10 (aswell as pAILo2 as a vector control) were used to transform Aspergillusoryzae JaL250 protoplasts. Eight transformants were isolated toindividual PDA plates and incubated for five days at 34° C. Confluentspore plates were washed with 5 ml of 0.01% TWEEN® 80 and the sporesuspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glassshake flasks. Transformant cultures were incubated at 34° C. withconstant shaking at 200 rpm. At day five post-inoculation, cultures werecentrifuged at 6000×g and their supernatants collected. Five microlitersof each supernatant were mixed with an equal volume of 2× loading buffer(10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-glycineSDS-PAGE gel and stained with SIMPLY BLUE™ SafeStain. SDS-PAGE profilesof the culture broths showed that eight out of eight transformants had anew protein band of approximately 50 kDa. Transformant number 8 wasselected for further studies and designated A. oryzae JaL250AILo10.

A new fully confluent spore plate was prepared as described above.Spores were collected with 5 ml of an aqueous solution of 0.01% TWEEN®80 and two more washes with MDU2BP medium to maximize the number ofspores collected. The spore suspension was then used to inoculate 500 mlof MDU2BP medium in a two-liter Fernbach flask. The A. oryzaeJaL250AILo10 liquid culture was then incubated at 34° C. with shaking at200 rpm. At day five post-inoculation the culture broth was collected byfiltration on a 500 milliliter, 75 mm Nylon filter unit with a pore sizeof 0.45 μm.

The culture filtrate was desalted and buffer exchanged in 20 mM Tris,150 mM NaCl pH 8.5, using a HIPREP® 26/10 desalting column (GEHealthcare, Piscataway, N.J., USA) according to the manufacturer'sinstructions Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 3: Preparation of Aspergillus fumigatus NN055679 Cel7ACellobiohydrolase I

A tfasty search (Pearson et al., 1997, Genomics 46:24-36) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was performed using as query a Cel7cellobiohydrolase protein sequence from Trichoderma reesei (AccessionNo. P00725). Several genes were identified as putative Family GH7homologs based upon a high degree of similarity to the query sequence atthe amino acid level. One genomic region with significant identity tothe query sequence was chosen for further study, and the correspondinggene was named cel7A.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify an Aspergillus fumigatus NN055679 cel7A cellobiohydrolase I gene(SEQ ID NO: 5 [DNA sequence] and SEQ ID NO: 6 [deduced amino acidsequence]) from genomic DNA of Aspergillus fumigatus prepared asdescribed in WO 2005/047499.

Forward primer: (SEQ ID NO: 79) 5′-gggcATGCTGGCCTCCACCTTCTCC-3′Reverse primer: (SEQ ID NO: 80) 5′-gggttaattaaCTACAGGCACTGAGAGTAA-3′

Upper case letters represent the coding sequence. The remainder of thesequence provides restriction endonuclease sites for Sph I and Pac I inthe forward and reverse sequences, respectively. Using these primers,the Aspergillus fumigatus cel7A gene was amplified using standard PCRmethods and the reaction product isolated by 1% agarose gelelectrophoresis using TAE buffer and purified using a QIAQUICK® GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions.

The fragment was digested with Sph I and Pac I and ligated into theexpression vector pAILo2 also digested with Sph I and Pac I according tostandard procedures. The ligation products were transformed into E. coliXL10 SOLOPACK® cells (Stratagene, La Jolla, Calif., USA) according tothe manufacturer's instructions. An E. coli transformant containing aplasmid of the correct size was detected by restriction digestion andplasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia,Calif., USA). DNA sequencing of the insert gene from this plasmid wasperformed with a Perkin-Elmer Applied Biosystems Model 377 XL AutomatedDNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City,Calif., USA) using dye-terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The nucleotide sequencewas shown to match the genomic sequence determined by TIGR (SEQ ID NO: 5[DNA sequence] and SEQ ID NO: 6 [deduced amino acid sequence]). Theresulting plasmid was named pEJG93.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Five μg of pEJG93 (as well aspAILo2 as a vector control) was used to transform Aspergillus oryzaeJaL250.

The transformation of Aspergillus oryzae JaL250 with pEJG93 yieldedabout 100 transformants. Ten transformants were isolated to individualPDA plates.

Confluent PDA plates of five of the ten transformants were washed with 5ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.Five days after incubation, 0.5 μl of supernatant from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that one of the transformants had amajor band of approximately 70 kDa. This transformant was namedAspergillus oryzae JaL250EJG93.

Five hundred ml of shake flask medium were added to a 2800 ml shakeflask. The shake flask medium was composed of 45 g of maltose, 2 g ofK2HPO₄, 12 g of KH₂PO₄, 1 g of NaCl, 1 g of MgSO₄.7H₂O, 7 g of yeastextract, 2 g of urea, and 0.5 ml of trace elements solution. The traceelements solution was composed per liter of 13.8 g of FeSO₄.7H₂O, 14.3 gof ZnSO₄.7H₂O, 8.5 g of MnSO₄. H₂O, 2.5 g of CuSO4.5H₂O, 0.5 g ofNiCl₂.6H₂O, 3 g of citric acid, and deionized water to 1 liter. Twoshake flasks were inoculated with a suspension of a PDA plate ofAspergillus oryzae JaL250EJG93 with 0.01% TWEEN® 80 and incubated at 34°C. on an orbital shaker at 200 rpm for 120 hours. The broth was filteredusing a 0.7 μm Whatman glass filter GF/F (Whatman, Piscataway, N.J.,USA) followed by a 0.22 μm EXPRESS™ Plus Membrane (Millipore, Bedford,Mass., USA).

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator (Pall Filtron, Northborough, Mass., USA) equipped witha 10 kDa polyethersulfone membrane (Pall Filtron, Northborough, Mass.,USA) with 20 mM Tris-HCl pH 8.5. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 4: Preparation of Thermoascus aurantiacus CGMCC 0583 Cel7ACellobiohydrolase I

The Thermoascus aurantiacus CGMCC 0583 Cel7A cellobiohydrolase I (CBHI)gene (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced amino acidsequence]) was isolated according to WO 2003/000941 and expressed inAspergillus oryzae JaL250.

The fungal strain Thermoascus aurantiacus CGMCC 0583 was grown on anagar plate composed of 0.5% yeast extract, 1% glucose, and 2% agar for 3days at 45° C. The fully grown culture was used to inoculate shakeflasks containing liquid medium composed of 3% soymeal, 1.5% maltose,and 0.5% peptone. The flasks were incubated at 45° C. for 48 hours withshaking. The mycelia were harvested by centrifugation of the culturebroth at 8000 rpm, 4° C. for 30 minutes, transferred into a cleanplastic bag followed by immediate freezing in liquid nitrogen, andstored at −80° C. before total RNA was isolated.

The frozen mycelia were grounded into a very fine powder with asterilized mortar and pestle baked at 200° C. for 24 hours. An RNEASY®Plant Mini Kit was used to isolate total RNA according to themanufacturer's instructions.

First strand cDNA synthesis from the total RNA was performed using a 3′RACE System for Rapid Amplification of cDNA Ends according to themanufacturer's instructions. The first strand cDNA of 3′ RACE was usedas PCR template for PCR screening.

Two oligonucleotides shown below were used for PCR screening of cDNA ofThermoascus aurantiacus CGMCC 0583. The forward primer was derived froman alignment of conserved regions of cellobiohydrolase I genes and thereverse primer was provided by the 3′ RACE System.

Forward primer: (SEQ ID NO: 81) 5′-GGnACnGGnTA(t/c)TG(t/c)GA-3′Reverse primer: (SEQ ID NO: 82) 5′-TCnA(a/g)CCAnA(a/g)CAT(a/g)TT-3′

One hundred picomoles of the above primers were used in a PCR reactioncomposed of 2 μl of the first strand cDNA of 3′ RACE, 5 μl of 10×Taq DNApolymerase buffer, 3 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTP, and 2.5units of Taq DNA polymerase in a final volume of 50 μl. Theamplification was performed in a thermocycler programmed for 1 cycle at95° C. for 3 minutes; 30 cycles each at 95° C. for 30 seconds, 50° C.for 30 seconds, and 72° C. for 50 seconds; and 1 cycle at 72° C. for 10minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 0.65 kb product band was excisedfrom the gel, and purified using a WIZARD® PCR Preps DNA PurificationSystem according to the manufacturer's instructions. The PCR product wassequenced using a 377 DNA Analyzer. Sequencing showed that the 0.65 kbfragment was homologous to cellobiohydrolase 1.

Two oligos were designed for 5′ end cloning of Thermoascus aurantiacusCGMCC 0583 Cel7A cellobiohydrolase 1 by using a 5′ RACE System for RapidAmplification of cDNA Ends.

Primer 025AS1: (SEQ ID NO: 83) 5′-GTAGAGATGCTGTTGGCT-3′ Primer 025AS1.5:(SEQ ID NO: 84) 5′-TCTCAGCGCAGCAGGAACCGT-3′

The gene specific primer 025AS1 was used for first strand cDNA synthesisusing the 5′ RACE System according to the manufacturer's instructions.The first strand cDNA of 5′ RACE was used as template for a PCRamplification composed of 5 μl of 10×Taq DNA polymerase buffer, 3 μl of25 mM MgCl₂, 1 μl of 10 mM dNTP, 2 μl of 10 μM primer 025AS1.5, 2 μl of10 μM primer AAP (Abridged Anchor Primer, provided by the kit), and 2.5units of Taq DNA polymerase in a final volume of 50 μl. Theamplification was performed in a thermocycler programmed for 1 cycle at94° C. for 2 minutes; 30 cycles each at 94° C. for 40 seconds, 55° C.for 40 seconds, and 72° C. for 60 seconds; and 1 cycle at 72° C. for 10minutes. The heat block then went to a 4° C. soak cycle.

The PCR products were isolated by 1.0% agarose gel electrophoresis usingTBE buffer and purified using a WIZARD® PCR Preps DNA PurificationSystem. A dominant DNA fragment at 0.8 kb was confirmed to be the 5′ endof Thermoascus aurantiacus CGMCC 0583 Cel7A cellobiohydrolase 1 gene bysequencing using a 377 DNA Analyzer.

One forward primer, 1F shown below, was designed based on the sequenceinformation of the 5′ end cloning. Primer 1F was used for thefull-length cloning of the Thermoascus aurantiacus CGMCC 0583 Cel7Acellobiohydrolase 1 gene together with primer AUAP (provided by the kit)as the reverse primer.

Primer 1F: (SEQ ID NO: 85) 5′-AGCGACAGCAATAACAAT-3′

Ten picomoles of the above 2 primers were used in a PCR reactioncomposed of the 4 μl of first strand cDNA of 3′ RACE, 5 μl of 10×Taq DNApolymerase buffer, 3 μl of 25 mM MgCl₂ 1 μl of 10 mM dNTP, and 2.5 unitsof Taq DNA polymerase in a final volume of 50 μl. The amplification wasperformed in a thermocycler programmed for 1 cycle at 95° C. for 2minutes; 30 cycles each at 95° C. for 40 seconds, 58° C. for 40 seconds,and 72° C. for 90 seconds; and 1 cycle at 72° C. for 10 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.7 kb product band was excisedfrom the gel, and purified using a WIZARD® PCR Preps DNA PurificationSystem. The PCR fragment was then ligated to pGEM-T using a pGEM-TVector System. The plasmid DNA was confirmed by sequencing using a 377DNA Analyzer. The correct clone was designated pT002-5.

Two synthetic oligonucleotide primers shown below containing Bsp LU11Isites on the forward primer and Pac I at the reverse primer weredesigned to PCR amplify the full-length open reading frame of theThermoascus aurantiacus CGMCC 0583 Cel7A cellobiohydrolase I gene. ARapid Ligation Kit was used to clone the fragment into pAILo2.

PCR Forward primer: (SEQ ID NO: 86) 5′-ACATGT ATCAGCGCGCTCTTCTC-3′PCR Reverse primer: (SEQ ID NO: 87) 5′-TTAATTAA TTAGTTGGCGGTGAAGGTCG-3′Bold letters represent coding sequence. The underlined sequence containssequence identity to the Bsp LU11I and Pac I restriction sites.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 50 ng of plasmid pT002-5, 1×Pwo Amplification Buffer withMgSO₄, 4 μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, and 2.5 unitsof Pwo DNA Polymerase, in a final volume of 50 μl. A DNA ENGINE™ ThermalCycler was used to amplify the fragment programmed for one cycle at 94°C. for 2 minutes; and 25 cycles each at 94° C. for 30 seconds, 59° C.for 30 seconds, and 72° C. for 1.5 minutes. After the 25 cycles, thereaction was incubated at 72° C. for 10 minutes and then cooled at 10°C. until further processed.

A 1.3 kb PCR reaction product was isolated on a 0.8% GTG® agarose gelusing TAE buffer and 0.1 μg of ethidium bromide per ml. The DNA band wasvisualized with the aid of a DARKREADER™ Transilluminator to avoidUV-induced mutations. The 1.3 kb DNA band was excised with a disposablerazor blade and purified with an ULTRAFREE® DA spin cup according to themanufacturer's instructions.

The purified PCR fragment was cloned into pCR®4Blunt-TOPO® according tothe manufacturer's instructions. PCR clones containing the coding regionof interest were sequenced to Phred Q values of at least 40 to insurethat there were no PCR induced errors. All sequence aligments wereperformed with Consed (University of Washington). One of the clones thatwas shown to have the expected sequence was selected and re-named TaPCR.The TaPCR clone containing the T. aurantiacus cellobiohydrolase I codingregion was double digested with the restriction enzymes Bsp LU11I andPac I and gel purified as described above. This DNA fragment was thenligated into pAILo2 previously digested with Nco I and Pac I using aRapid Ligation Kit. Expression clones were confirmed by restrictiondigestion and sequenced to confirm that the junction vector-insert wascorrect. Plasmid DNA for transformation was prepared with a Midi-PrepKit. The final clone was re-named pAILo6.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Ten micrograms of pAILo6 wereused to transform the Aspergillus oryzae JaL250 protoplasts. Twelvetransformants were isolated to individual PDA plates and incubated for 5days at 34° C. Confluent spore plates were washed with 5 ml of 0.01%TWEEN® 80 and the spore suspension was used to inoculate 25 ml of MDU2BPmedium in 125 ml glass shake flasks. Transformant cultures wereincubated at 34° C. with constant shaking at 200 rpm. At day fivepost-inoculation, cultures were centrifuged at 6000×g and theirsupernatants collected. Five microliters of each supernatant were mixedwith an equal volume of 2× loading buffer (10% beta-mercaptoethanol) andanalyzed by SDS-PAGE using a 1.5 mm 8%-16% Tris-glycine SDS-PAGE gel andstained with SIMPLY BLUE™ SafeStain. SDS-PAGE profiles of the culturebroths showed that eleven out of twelve transformants had a new proteinband of approximately 60 kDa. Transformant number 12 was selected forfurther studies and designated A. oryzae JaL250AILo6.

A spore stock suspension was prepared from a 5 day plate culture of A.oryzae JaL250AILo6 by adding 10 ml of 0.1% TWEEN® 20 to the cultureplate to release the spores. A shake flask culture was started byinoculating 100 μl of the spore stock to a 250 ml baffled flaskcontaining 50 ml of M410 medium pH 6.0. The shake flask was grown at 34°C. for 5 days with shaking a 250 rpm. The culture was filtered through a0.2 μm pore filter device and the filtrate was recovered for proteinpurification.

A 50 ml volume of the filtrate was desalted and buffer exchanged in 20mM sodium acetate pH 5.0 using an ECONO-PAC® 10-DG desalting columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 5: Preparation of Myceliophthora thermophila CBS 117.65 Cel6ACellobiohydrolase II

The Myceliophthora thermophila CBS 117.65 Cel6A cellobiohydrolase II(SEQ ID NO: 9 [DNA sequence] and SEQ ID NO: 10 [deduced amino acidsequence]) was obtained according to the procedure described below.

One hundred ml of shake flask medium was added to a 500 ml shake flask.The shake flask medium was composed per liter of 15 g of glucose, 4 g ofK₂HPO₄, 1 g of NaCl, 0.2 g of MgSO₄.7H₂O, 2 g of MES free acid, 1 g ofBacto Peptone, 5 g of yeast extract, 2.5 g of citric acid, 0.2 g ofCaCl₂.2H₂O, 5 g of NH₄NO₃, and 1 ml of trace elements solution. Thetrace elements solution was composed per liter of 1.2 g of FeSO₄.7H₂O,10 g of ZnSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.4 g of CuSO₄.5H₂O, 0.4 g ofNa₂B₄O₇.10H₂O, and 0.8 g of Na₂MoO₂.2H₂O. The shake flask was inoculatedwith two plugs from a solid plate culture of Myceliophthora thermophilastrain CBS 117.65 and incubated at 45° C. with shaking at 200 rpm for 48hours. Fifty ml of the shake flask broth was used to inoculate a 2 literfermentation vessel.

Fermentation batch medium was composed per liter of 5 g of yeastextract, 176 g of powdered cellulose, 2 g of glucose, 1 g of NaCl, 1 gof Bacto Peptone, 4 g of K2HPO₄, 0.2 g of CaCl₂.2H₂O, 0.2 g ofMgSO₄.7H₂O, 2.5 g of citric acid, 5 g of NH₄NO₃, 1.8 ml of anti-foam,and 1 ml of trace elements solution (above). Fermentation feed mediumwas composed of water and antifoam.

A total of 1.8 liters of the fermentation batch medium was added to atwo liter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). Fermentation feed medium was dosed at a rate of 4 g/l/hrfor a period of 72 hours. The fermentation vessel was maintained at atemperature of 45° C. and pH was controlled using an Applikon 1030control system (Applikon Biotechnology, Schiedam, Netherlands) to aset-point of 5.6+/−0.1. Air was added to the vessel at a rate of 1 vvmand the broth was agitated by Rushton impeller rotating at 1100 to 1300rpm. At the end of the fermentation, whole broth was harvested from thevessel and centrifuged at 3000×g to remove the biomass.

The harvested broth obtained above was centrifuged in 500 ml bottles at13,000×g for 20 minutes at 4° C. and then sterile filtered using a 0.22μm polyethersulfone membrane (Millipore, Bedford, Mass., USA). Thefiltered broth was concentrated and buffer exchanged with 20 mM Tris-HClpH 8.5 using a tangential flow concentrator equipped with a 10 kDapolyethersulfone membrane at approximately 20 psi. To decrease theamount of pigment, the concentrate was applied to a 60 ml Q SEPHAROSE™Big Bead column equilibrated with 20 mM Tris-HCl pH 8.5, and step elutedwith equilibration buffer containing 600 mM NaCl. Flow-through andeluate fractions were examined on 8-16% CRITERION® SDS-PAGE gels(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) stained withGELCODE® Blue Stain Reagent (Bio-Rad Laboratories, Inc., Hercules,Calif., USA). The flow-through fraction contained the Myceliophthorathermophila Cel6A cellobiohydrolase as judged by the presence of a bandcorresponding to the apparent molecular weight of the protein bySDS-PAGE (approximately 75 kDa).

The flow-through fraction was concentrated using an ultrafiltrationdevice (Millipore, Bedford, Mass., USA) equipped with a 10 kDapolyethersulfone membrane at 40 psi, 4° C. and mixed with an equalvolume of 20 mM Tris-HCl pH 7.5 containing 3.4 M ammonium sulfate for afinal concentration of 1.7 M ammonium sulfate. The sample was filtered(0.2 μM syringe filter, polyethersulfone membrane, Whatman, Maidstone,United Kingdom) to remove particulate matter prior to loading onto aPHENYL SUPEROSE™ column (HR 16/10, GE Healthcare, Piscataway, N.J., USA)equilibrated with 1.7 M ammonium sulfate in 20 mM Tris-HCl pH 7.5. Boundproteins were eluted with a 12 column volume decreasing salt gradient of1.7 M ammonium sulfate to 0 M ammonium sulfate in 20 mM Tris-HCl pH 7.5.Fractions were analyzed by 8-16% SDS-PAGE gel electrophoresis asdescribed above, which revealed that the Myceliophthora thermophilaCel6A cellobiohydrolase eluted at the very end of the gradient(approximately 20 mM ammonium sulfate).

Fractions containing the Cel6A cellobiohydrolase II were pooled anddiluted 10-fold in 20 mM Tris-HCl pH 9.0 (to lower the salt and raisethe pH) and then applied to a 1 ml RESOURCE™ Q column (GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 9.0. Boundproteins were eluted with a 20 column volume salt gradient from 0 mM to550 mM NaCl in 20 mM Tris-HCl pH 9.0. M. thermophila Cel6Acellobiohydrolase II eluted as a single peak early in the gradient (˜25mM NaCl). The cellobiohydrolase II was >90% pure as judged by SDS-PAGE.Protein concentrations were determined using a BCA Protein Assay Kit(Pierce, Rockford, Ill., USA) in which bovine serum albumin was used asa protein standard.

Example 6: Preparation of Recombinant Myceliophthora thermophila CBS202.75 Cel6A Cellobiohydrolase II

Myceliophthora thermophila CBS 202.75 was grown in 100 ml of YEG mediumin a baffled shake flask at 45° C. for 2 days with shaking at 200 rpm.Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, LaJolla, Calif., USA), washed twice in deionized water, and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN Inc., Valencia, Calif., USA).

A full-length Family 6 cellobiohydrolase gene (Cel6A) was isolated fromMyceliophthora thermophila CBS 202.75 using a GENOMEWALKER™ UniversalKit (Clontech Laboratories, Inc., Mountain View, Calif., USA) accordingto the manufacturer's instructions. Briefly, total genomic DNA fromMyceliophthora thermophila CBS 202.75 was digested separately with fourdifferent restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) thatleave blunt ends. Each batch of digested genomic DNA was then ligatedseparately to the GENOMEWALKER™ Adaptor (Clontech Laboratories, Inc.,Mountain View, Calif., USA) to create four libraries. These librarieswere then employed as templates in PCR reactions using two gene-specificprimers shown below, one for primary PCR and one for secondary PCR. Theprimers were designed based on a partial Family 6 cellobiohydrolase gene(Cel6A) sequence from Myceliophthora thermophila (WO 2004/056981).

Primer MtCel6A-R4: (SEQ ID NO: 88) 5′-ATTGGCAGCCCGGATCTGGGACAGAGTCTG-3′Primer MtCel6A-R5: (SEQ ID NO: 89) 5′-CCGGTCATGCTAGGAATGGCGAGATTGTGG-3′

The primary amplifications were composed of 1 μl (approximately 6 ng) ofeach library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10μmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View,Calif., USA), 10 μmol of primer MtCel6A-R4, 1× ADVANTAGE® GC-Melt LABuffer (Clontech Laboratories, Inc., Mountain View, Calif., USA), and1.25 units of ADVANTAGE® GC Genomic Polymerase Mix (ClontechLaboratories, Inc., Mountain View, Calif., USA) in a final volume of 25μl. The amplification reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for pre-denaturing at 94° C. for 1 minute; 7 cycles each at adenaturing temperature of 94° C. for 30 seconds; annealing andelongation at 72° C. for 5 minutes; and 32 cycles each at 67° C. for 5minutes.

The secondary amplifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 μmolof Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif.,USA), 10 μmol of primer MtCel6A-R5, lx ADVANTAGE® GC-Melt LA Buffer, and1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of25 μl. The amplifications were incubated in an EPPENDORF® MASTERCYCLER®5333 programmed for pre-denaturing at 94° C. for 1 minute; 5 cycles eachat a denaturing temperature of 94° C. for 30 seconds; annealing andelongation at 72° C. for 5 minutes; and 20 cycles at 67° C. for 5minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 3.5 kb product band from the Eco RV library wasexcised from the gel, purified using a QIAQUICK® Gel Extraction Kitaccording to the manufacturer's instructions, and sequenced.

DNA sequencing of the 3.5 kb PCR fragment was performed with aPerkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. The following genespecific primers were used for sequencing:

MtCel6A-F2: (SEQ ID NO: 90) 5′-GCTGTAAACTGCGAATGGGTTCAG-3′ MtCel6A-F3:(SEQ ID NO: 91) 5′-GGGTCCCACATGCTGCGCCT-3′ MtCel6A-R8: (SEQ ID NO: 92)5′-AAAATTCACGAGACGCCGGG-3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The 3.5 kb sequence wascompared and aligned with a partial Family 6 cellobiohydrolase gene(Cel6A) sequence from Myceliophthora thermophila (WO 2004/056981).

A gene model for the Myceliophthora thermophila sequence was constructedbased on similarity of the encoded protein to homologous glycosidehydrolase Family 6 proteins from Thielavia terrestris, Chaetomiumthermophilum, Humicola insolens, and Trichoderma reesei. The nucleotidesequence and deduced amino acid sequence of the Myceliophthorathermophila CBS 202.75 Cel6A cellobiohydrolase II gene are shown in SEQID NO: 11 and SEQ ID NO: 12, respectively. The genomic fragment encodesa polypeptide of 482 amino acids, interrupted by 3 introns of 96, 87,and 180 bp. The % G+C content of the gene and the mature coding sequenceare 61.6% and 64%, respectively. Using the SignalP software program(Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal peptide of17 residues was predicted. The predicted mature protein contains 465amino acids with a molecular mass of 49.3 kDa.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Myceliophthora thermophila cellobiohydrolase gene from thegenomic DNA prepared above for construction of an Aspergillus oryzaeexpression vector. An IN-FUSION™ Cloning Kit (BD Biosciences, Palo Alto,Calif., USA) was used to clone the fragment directly into the expressionvector pAILo2, without the need for restriction digestion and ligation.

MtCel6A-F4: (SEQ ID NO: 93) 5′-ACTGGATTTACCATGGCCAAGAAGCTTTTCATCACC-3′MtCel6A-R9: (SEQ ID NO: 94) 5′-TCACCTCTAGTTAATTAATTAGAAGGGCGGGTTGGCGT-3′

Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Myceliophthora thermophila genomic DNA, 1×ADVANTAGE® GC-Melt LA Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP,and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volumeof 25 μl. The amplification reaction was incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 94° C. for 1 minutes; and30 cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72°C. for 2 minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1842 bp product band was excised from the gel,and purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

Plasmid pAILo2 was digested with Nco I and Pac I, isolated by 1.0%agarose gel electrophoresis using TAE buffer, and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Cloning Kit resulting in pSMai180 in which transcription ofthe cellobiohydrolase gene was under the control of a NA2-tpi promoter(a modified promoter from the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans). The ligation reaction (50 μl) was composed of1× IN-FUSION™ Buffer (BD Biosciences, Palo Alto, Calif., USA), 1×BSA (BDBiosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION™ enzyme (diluted1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng of pAILo2digested with Nco I and Pac I, and 50 ng of the Myceliophthorathermophila Cel6A purified PCR product. The reaction was incubated atroom temperature for 30 minutes. One μl of the reaction was used totransform E. coli XL10 SOLOPACK® Gold cells. An E. coli transformantcontaining pSMai180 was detected by restriction digestion and plasmidDNA was prepared using a BIOROBOT® 9600. The Myceliophthora thermophilaCel6A insert in pSMai180 was confirmed by DNA sequencing.

The same 1842 bp PCR fragment was cloned into pCR®2.1-TOPO® (Invitrogen,Carlsbad, Calif., USA) using a TOPO® TA CLONING® Kit to generatepSMai182. The Myceliophthora thermophila cel6A gene insert in pSMai182was confirmed by DNA sequencing. E. coli pSMai182 was deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, III., 61604,on Sep. 6, 2007.

The Myceliophthora thermophila Family 6 cellobiohydrolase Cel6A gene wasexpressed in Aspergillus oryzae JaL355. A. oryzae JaL355 (WO 2002/40694)protoplasts were prepared according to the method of Christensen et al.,1988, supra. Three μg of pSMai180 were used to transform Aspergillusoryzae JaL355.

The transformation of Aspergillus oryzae JaL355 with pSMai180 yieldedabout 50 transformants. Twenty transformants were isolated to individualMinimal medium plates.

Confluent Minimal Medium plates of the 20 transformants were washed with5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed using 8-16% CRITERION® SDS-PAGE gels and a CRITERION® Cell(Bio-Rad Laboratories, Inc., Hercules, Calif., USA), according to themanufacturer's instructions. The resulting gel was stained withBIO-SAFE™ Coomassie Stain (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 70 kDa.

A confluent plate of one transformant, designated transformant 14, waswashed with 10 ml of 0.01% TWEEN® 20 and inoculated into a 2 literFernbach flask containing 500 ml of MDU2BP medium to generate broth forcharacterization of the enzyme. The culture was harvested on day 5 andfiltered using a 0.22 μm EXPRESS™ Plus Membrane.

The filtered broth was concentrated and buffer exchanged using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane with 20 mM Tris-HCl pH 8.0. The concentrated and bufferexchanged broth was adjusted to 20 mM Tris-HCl pH 8.0-1.2 M (NH₄)₂SO₄and applied to a Phenyl SUPEROSE™ column (HR 16/10) equilibrated with 20mM Tris-HCl pH 8.0-1.2 M (NH₄)₂SO₄. Bound proteins were eluted with alinear gradient over 10 column volumes from 300 to 0 mM (NH₄)₂SO₄ in 20mM Tris-HCl pH 8.0. SDS-PAGE of eluate fractions showed a major band atapproximately 70 kDa. These fractions were then concentrated and bufferexchanged by centrifugal concentration using a VIVASPIN™ centrifugalconcentrator (10 kDa polyethersulfone membrane, Sartorius, Göttingen,Germany) into 20 mM Tris-HCl pH 8.0. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 7: Preparation of Thielavia terrestris NRRL 8126 Cel6ACellobiohydrolase II (CBHII)

Thielavia terrestris NRRL 8126 Cel6A cellobiohydrolase II (SEQ ID NO: 13[DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2006/074435 using Trichodermareesei as a host.

Culture filtrate was desalted and buffer exchanged in 20 mM Tris-150 mMsodium chloride pH 8.5 using an ECONO-PAC® 10-DG desalting columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 8: Preparation of Trichophaea saccata CBS 804.70Cellobiohydrolase II (CBHII)

The Trichophaea saccata CBS 804.70 cellobiohydrolase II (CBHII) (SEQ IDNO: 15 [DNA sequence] and SEQ ID NO: 16 [deduced amino acid sequence])was prepared as described below.

Trichophaea saccata CBS 804.70 was inoculated onto a PDA plate andincubated for 7 days at 28° C. Several mycelia-PDA agar plugs wereinoculated into 750 ml shake flasks containing 100 ml of MEX-1 medium.The flasks were incubated at 37° C. for 9 days with shaking at 150 rpm.The fungal mycelia were harvested by filtration through MIRACLOTH®(Calbiochem, San Diego, Calif., USA) before being frozen in liquidnitrogen. The mycelia were then pulverized into a powder by milling thefrozen mycelia together with an equal volume of dry ice in a coffeegrinder precooled with liquid nitrogen. The powder was transferred intoa liquid nitrogen prechilled mortar and pestle and ground to a finepowder with a small amount of baked quartz sand. The powdered mycelialmaterial was kept at −80° C. until use.

Total RNA was prepared from the frozen, powdered mycelia of Trichophaeasaccata CBS 804.70 by extraction with guanidium thiocyanate followed byultracentrifugation through a 5.7 M CsCl cushion according to Chirgwinet al., 1979, Biochemistry 18: 5294-5299. The polyA enriched RNA wasisolated by oligo (dT)-cellulose affinity chromatography according toAviv et al., 1972, Proc. Natl. Acad. Sci. USA 69: 1408-1412.

Double stranded cDNA was synthesized according to the general methods ofGubler and Hoffman, 1983, Gene 25: 263-269; Sambrook, J., Fritsch, E.F., and Maniantis, T. Molecular cloning: A Laboratory Manual, 2^(nd)ed., 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andKofod et al., 1994, J. Biol. Chem. 269: 29182-29189, using a polyA-Not Iprimer (Promega Corp., Madison, Wis., USA). After synthesis, the cDNAwas treated with mung bean nuclease, blunt ended with T4 DNA polymerase,and ligated to a 50-fold molar excess of Eco RI adaptors (InvitrogenCorp., Carlsbad, Calif., USA). The cDNA was cleaved with Not I and thecDNA was size fractionated by 0.8% agarose gel electrophoresis using in44 mM Tris base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer. Thefraction of cDNA of 700 bp and larger was excised from the gel andpurified using a GFX® PCR DNA and Gel Band Purification Kit (GEHealthcare Life Sciences, Piscataway, N.J., USA) according to themanufacturer's instructions.

The prepared cDNA was then directionally cloned by ligation into EcoRI-Not I cleaved pMHas5 (WO 03/044049) using a Rapid Ligation Kit (RocheDiagnostics GmbH, Penzberg, Germany) according to the manufacturer'sinstructions. The ligation mixture was electroporated into E. coli DH10Bcells (Invitrogen Corp., Carlsbad, Calif., USA) using a GENE PULSER® andPulse Controller (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at50 μF, 25 mAmp, 1.8 kV with a 2 mm gap cuvette according to themanufacturer's procedure.

The electroporated cells were spread onto LB plates supplemented with 50μg of kanamycin per ml. A cDNA plasmid pool was prepared fromapproximately 30,000 total transformants of the original cDNA-pMHas5vector ligation. Plasmid DNA was prepared directly from the pool ofcolonies using a QIAPREP® Spin Midi/Maxiprep Kit (QIAGEN GmbHCorporation, Hilden, Germany). The cDNA library was designated SBL521-2.

A transposon containing plasmid designated pSigA4 was constructed fromthe pSigA2 transposon containing plasmid described in WO 01/77315 inorder to create an improved version of the signal trapping transposon ofpSigA2 with decreased selection background. The pSigA2 transposoncontains a signal less beta-lactamase construct encoded on thetransposon itself. PCR was used to create a deletion of the intactbeta-lactamase gene found on the plasmid backbone using a proofreadingPROOFSTART® DNA polymerase (QIAGEN GmbH Corporation, Hilden, Germany)and the following 5′ phosphorylated primers (TAG Copenhagen, Denmark):

SigA2NotU-P: (SEQ ID NO: 95) 5′-TCGCGATCCGTTTTCGCATTTATCGTGAAACGCT-3′SigA2NotD-P: (SEQ ID NO: 96) 5′-CCGCAAACGCTGGTGAAAGTAAAAGATGCTGAA-3′

The amplification reaction was composed of 1 μl of pSigA2 (10 ng/μl), 5μl of 10× ProofStart Buffer (QIAGEN GmbH Corporation, Hilden, Germany),2.5 μl of dNTP mix (20 mM), 0.5 μl of SigA2NotU-P (10 mM), 0.5 μl ofSigA2NotD-P (10 mM), 10 μl of Q solution (QIAGEN GmbH Corporation,Hilden, Germany), and 31.25 μl of deionized water. A DNA ENGINE™ ThermalCycler (MJ Research Inc., Waltham, Mass., USA) was used for theamplification programmed for 1 cycle at 95° C. for 5 minutes; and 20cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for 4 minutes.

A 3.9 kb PCR reaction product was isolated by 0.8% agarose gelelectrophoresis using TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of an Eagle Eye Imaging System(Stratagene, La Jolla, Calif., USA) at 360 nm. The 3.9 kb DNA band wasexcised from the gel and purified by using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 3.9 kb fragment was self-ligated at 16° C. overnight with 10 unitsof T4 DNA ligase (New England Biolabs, Inc., Beverly, Mass., USA), 9 μlof the 3.9 kb PCR fragment, and 1 μl of 10× ligation buffer (New EnglandBiolabs, Inc., Beverly, Mass., USA). The ligation was heat inactivatedfor 10 minutes at 65° C. and then digested with Dpn I at 37° C. for 2hours. After incubation, the digestion was purified using a GFX® PCR DNAand Gel Band Purification Kit.

The purified material was then transformed into E. coli TOP10 competentcells (Invitrogen Corp., Carlsbad, Calif., USA) according to themanufacturer's instructions. The transformation mixture was plated ontoLB plates supplemented with 25 μg of chloramphenicol per ml. Plasmidminipreps were prepared from several transformants and digested with BglII. One plasmid with the correct construction was chosen. The plasmidwas designated pSigA4. Plasmid pSigA4 contains the Bgl II flankedtransposon SigA2 identical to that disclosed in WO 01/77315.

A 60 μl sample of plasmid pSigA4 DNA (0.3 μg/μl) was digested with BglII and separated by 0.8% agarose gel electrophoresis using TAE buffer. ASigA2 transposon DNA band of 2 kb was eluted with 200 μl of EB buffer(QIAGEN GmbH Corporation, Hilden, Germany) and purified using a GFX® PCRDNA and Gel Band Purification Kit according to the manufacturer'sinstructions and eluted in 200 μl of EB buffer. SigA2 was used fortransposon assisted signal trapping.

A complete description of transposon assisted signal trapping can befound in WO 01/77315. A cDNA plasmid pool was prepared from 30,000 totaltransformants of the original cDNA-pMHas5 vector ligation. Plasmid DNAwas prepared directly from a pool of colonies recovered from solid LBselective medium using a QIAPREP® Spin Midi/Maxiprep Kit. The plasmidpool was treated with transposon SigA2 and MuA transposase (FinnzymesOY, Espoo, Finland) according to the manufacturer's instructions.

For in vitro transposon tagging of the Trichophaea saccata CBS 804.70cDNA library, 4 or 8 μl of SigA2 transposon containing approximately 2.6μg of DNA were mixed with 1 μl of the plasmid DNA pool of theTrichophaea saccata CBS 804.70 cDNA library containing 2 μg of DNA, 2 μlof MuA transposase (0.22 μg/μl), and 5 μl of 5× buffer (Finnzymes OY,Espoo, Finland) in a total volume of 50 μl and incubated at 30° C. for3.5 hours followed by heat inactivation at 75° C. for 10 minutes. TheDNA was precipitated by addition of 5 μl of 3 M sodium acetate pH 5 and110 μl of 96% ethanol and centrifuged for 30 minutes at 10,000×g. Thepellet was washed in 70% ethanol, air dried at room temperature, andresuspended in 10 μl of 10 mM Tris, pH 8, 1 mM EDTA (TE) buffer.

A 1.5 μl volume of the transposon tagged plasmid pool was electroporatedinto 20 μl of E. coli DH10B ultracompetent cells (Gibco-BRL,Gaithersburg Md., USA) according to the manufacturer's instructionsusing a GENE PULSER® and Pulse Controller (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) at 50 uF, 25 mAmp, 1.8 kV with a 2 mm gap cuvetteaccording to the manufacturer's procedure.

The electroporated cells were incubated in SOC medium with shaking at250 rpm for 2 hours at 28° C. before being plated on the followingselective media: LB medium supplemented with 50 μg of kanamycin per ml;LB medium supplemented with 50 μg of kanamycin per ml and 15 μg ofchloramphencol per ml; and/or LB medium supplemented with 50 μg ofkanamycin per ml, 15 μg of chloramphencol per ml, and 12.5 μg ofampicillin per ml.

From dilution plating of the electroporation onto LB medium supplementedwith kanamycin and chloramphencol medium, it was determined thatapproximately 72,000 colonies were present containing a cDNA libraryplasmid with a SigA2 transposition per electroporation and thatapproximately 69 colonies were recovered under triple selection (LB,kanamycin, chorlamphenicol, ampicillin). Further electroporation andplating experiments were performed until 445 total colonies wererecovered under triple selection. The colonies were miniprepped using aQIAPREP® 96 Turbo Miniprep Kit (QIAGEN GmbH Corporation, Hilden,Germany). Plasmids were sequenced with the transposon forward andreverse primers (primers A and B), shown below, according to theprocedure disclosed in WO 2001/77315 (page 28)

Primer A: (SEQ ID NO: 97) 5′-AGCGTTTGCGGCCGCGATCC-3′ Primer B:(SEQ ID NO: 98) 5′-TTATTCGGTCGAAAAGGATCC-3′

The Trichophaea saccata Family GH6 cDNA encoding cellobiohydrolase wassubcloned into the Aspergillus expression vector pMStr57 (WO2004/032648) by PCR amplifying the protein coding sequence from the cDNAlibrary SBL0521, described above, with the two synthetic oligonucleotideprimers shown below.

Primer 848: (SEQ ID NO: 99)5′-ACACAACTGGGGATCCTCATCATGAAGAACTTCCTTCTGG-3′ Primer 849:(SEQ ID NO: 100) 5′-CCCTCTAGATCTCGAGTTACGTGAAGCTAGGATTAGCATT-3′

The amplification was performed using IPROOF™ High Fidelity 2× MasterMix (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) following themanufacturer's instructions. The amplification reaction was composed ofSBL0521 pool DNA as template, 25 μmol each of primers 848 and 849, and25 μl of IPROOF™ High Fidelity 2× Master Mix in a final volume of 50 μl.The amplification was performed by pre-denaturing at 98° C. for 2minutes; 5 cycles each with denaturing at 98° C. for 10 seconds,annealing at 65° C. for 10 seconds, and elongation at 72° C. for 1minute; and 25 cycles each with denaturing at 98° C. for 10 seconds, andcombined annealing extension at 72° C. for 1 minute. A final elongationwas made at 72° C. for 10 minutes.

A PCR product of 1.4 kb was separated from residual reaction componentsusing a GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The PCR fragment was cloned into Barn HI and Xho I digested pMStr57using an IN-FUSION™ Dry-Down PCR Cloning Kit (Clontech Laboratories,Inc., Mountain View, Calif., USA). Approximately 50 ng of PCR productand 200 ng of vector in a total volume of 10 μl were added to theIN-FUSION™ Dry-Down pellet. The reaction was performed according to themanufacturer's instructions. The Trichophaea saccata Family GH6cellobiohydrolase encoding DNA of the resulting Aspergillus expressionconstruct, pMStr179, was sequenced and the sequence agreed completelywith the cellobiohydrolase coding sequence of SEQ ID NO: 16.

The same PCR fragment was cloned into the pCR®-BluntII-TOPO vector(Invitrogen, Life Technologies, Carlsbad, Calif., USA) using a ZeroBlunt TOPO PCR Cloning Kit, to generate pMStr199. The Trichophaeasaccata Family GH6 cellobiohydrolase encoding DNA of pMStr199 wassequenced and the sequence agreed completely with the cellobiohydrolasecoding sequence of SEQ ID NO: 1. E. coli strain NN059165, containingpMStr199, was deposited with the Deutsche Sammlung von Mikroorganismenand Zellkulturen GmbH (DSM), Braunschweig, Germany, on Feb. 24, 2010 andassigned the accession number DSM 23379.

The nucleotide sequence and deduced amino acid sequence of theTrichophaea saccata cellobiohydrolase cDNA are shown in SEQ ID NO: 1 andSEQ ID NO: 2, respectively. The coding sequence is 1344 bp including thestop codon. The encoded predicted protein is 447 amino acids. Using theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), asignal peptide of 16 residues was predicted. The predicted matureprotein contains 431 amino acids with a predicted molecular mass of 45.3kDa and an isoelectric pH of 5.06.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Trichophaea saccata cDNA encoding a Family GH6polypeptide having cellobiohydrolase activity shares 64% identity(excluding gaps) to the deduced amino acid sequence of acellobiohydrolase from Aspergillus fumigatus (GENESEQP:ABB80166).

The Aspergillus oryzae strain BECh2 (WO 2000/39322) was transformed withpMStr179 according to Christensen et al., 1988, Biotechnology 6,1419-1422 and WO 2004/032648. Ten transformants were cultured for 4 daysat 30° C. in 750 μl of DAP2C-1 medium (WO 2004/032648), in which 2%glucose was substituted for maltodextrin. Samples were monitored bySDS-PAGE using a CRITERION™ XT Precast 12% Bis-Tris gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. LMW standards from an Amersham LowMolecular Weight Calibration Kit for SDS Electrophoresis (GE HealthcareUK Limited, Buckinghamshire, UK) were used as molecular weight markers.The gel was stained with INSTANTBLUE™ (Expedeon Protein Solutions,Cambridge, UK). Eight transformants produced a novel protein doublet inthe range of 55-60 kDa.

Two of these transformants, designated Aspergillus oryzae MStr335 andMStr336, were isolated twice by dilution streaking conidia on selectivemedium (amdS) containing 0.01% TRITON® X-100 to limit colony size.

Spores from four confluent COVE N slants of Aspergillus oryzae MStr335spores were collected with a solution of 0.01% TWEEN® 20 and used toinoculate 21 shake flasks each containing 150 ml of DAP2C-1 medium (WO2004/032648) in which 2% glucose was substituted for maltodextrin. Theflasks were incubated at 30° C. with constant shaking at 200 rpm for 3days. Fungal mycelia and spores were removed at harvesting by firstfiltering the fermentation broth through a sandwich of 3 glassmicrofiber filters with increasing particle retention sizes of 1.6 μm,1.2 μm and 0.7 μm, and then filtering through a 0.45 μm filter. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 9: Preparation of Aspergillus fumigatus Cellobiohydrolase II

Aspergillus fumigatus NN055679 cellobiohydrolase II (CBHII) (SEQ ID NO:17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid sequence]) wasprepared according to the following procedure.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the full-length open reading frame of the Aspergillus fumigatusFamily 6A glycosyl hydrolase from genomic DNA. A TOPO Cloning kit wasused to clone the PCR product. An IN-FUSION™ Cloning Kit was used toclone the fragment into pAILo2.

Forward primer: (SEQ ID NO: 101)5′-ACTGGATTTACCATGAAGCACCTTGCATCTTCCATCG-3′ Reverse primer:(SEQ ID NO: 102) 5′-TCACCTCTAGTTAATTAAAAGGACGGGTTAGCGT-3′Bold letters represent coding sequence. The remaining sequence containssequence identity compared with the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 500 ng of Aspergillus fumigatus genomic DNA, 1× ThermoPol Taqreaction buffer (New England Biolabs, Ipswich, Mass., USA), 6 μl of 10mM blend of dATP, dTTP, dGTP, and dCTP, 0.1 unit of Taq DNA Polymerase(New England Biolabs, Ipswich, Mass., USA), in a final volume of 50 μl.An EPPENDORF® MASTERCYCLER® 5333 was used to amplify the fragmentprogrammed for one cycle at 98° C. for 2 minutes; and 35 cycles each at96° C. for 30 seconds, 61° C. for 30 seconds, and 72° C. for 2 minutes.After the 35 cycles, the reaction was incubated at 72° C. for 10 minutesand then cooled at 10° C. until further processed. To remove the A-tailsproduced by Taq the reaction was incubated for 10 minutes at 68° C. inthe presence of 1 unit of Pfx DNA polymerase (Invitrogen, Carlsbad,Calif., USA).

A 1.3 kb PCR reaction product was isolated on a 0.8% GTG-agarose gel(Cambrex Bioproducts, East Rutherford, N.J., USA) using TAE buffer and0.1 μg of ethidium bromide per ml. The DNA band was visualized with theaid of a DARK READER™ (Clare Chemical Research, Dolores, Colo.) to avoidUV-induced mutations. The 1.3 kb DNA band was excised with a disposablerazor blade and purified with an Ultrafree-DA spin cup (Millipore,Billerica, Mass.) according to the manufacturer's instructions.

The purified 1.3 kb PCR product was cloned into the PCR4Blunt-TOPOvector (Invitrogen). Two microliters of the purified PCR product weremixed with one microliter of a 2M Sodium chloride solution and onemicroliter of the Topo vector. The reaction was incubated at roomtemperarature for 15 minutes and then two microliters of the Toporeaction were used to transform E. coli TOP10 competent cells accordingto the manufacturer's instructions. Two aliquots of 100 microliters eachof the transformation reaction were spreaded onto two 150 mm 2xYT-Ampplates and incubated overnight at 37° C.

Eight recombinant colonies were used to inoculate liquid culturescontaining three milliliters of LB supplemented with 100 μg ofampicillin per milliliter of media. Plasmid DNA was prepared from thesecultures using a BIOROBOT® 9600. Clones were analyzed by restrictiondigest. Plasmid DNA from each clone was digested with the enzyme Eco RIaccording to the manufacturer instructions (NEB, Ipswich, Mass., USA)and analyzed by agarose gel electrophoresis as above. Six out of eightclones had the expected restriction digest pattern from these, clones 2,4, 5, 6, 7 and 8 were selected to be sequenced to confirm that therewere no mutations in the cloned insert. Sequence analysis of their5-prime and 3-prime ends indicated that clones 2, 6 and 7 had thecorrect sequence. These three clones were selected for re-cloning intopAILo2. One microliter aliquot of each clone was mixed with 17 μl ofdiluted TE (1:10 dilution) and 1 μl of this mix was used to re-amplifythe Aspergillus fumigatus glycosyl hydrolase 6A coding region.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μl of the diluted mix of clones 2, 6 and 7, 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of 10 mMblend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNAPolymerase, 1 μl of 50 mM MgSO4, in a final volume of 50 μl. AnEPPENDORF® MASTERCYCLER® 5333 was used to amplify the fragmentprogrammed for one cycle at 98° C. for 2 minutes; and 35 cycles each at94° C. for 30 seconds, 61° C. for 30 seconds, and 68° C. for 1.5minutes. After the 35 cycles, the reaction was incubated at 68° C. for10 minutes and then cooled at 10° C. until further processed. A 1.3 kbPCR reaction product was isolated on a 0.8% GTG-agarose gel using TAEbuffer and 0.1 μg of ethidium bromide per ml. The DNA band wasvisualized with the aid of a DARKREADER™ Transilluminator to avoidUV-induced mutations. The 1.0 kb DNA band was excised with a disposablerazor blade and purified with an Ultrafree-DA spin cup (Millipore,Billerica, Mass.) according to the manufacturer's instructions.

The vector pAILo2 was linearized by digestion with Nco I and Pac I(using conditions specified by the manufacturer). The fragment waspurified by gel electrophoresis and ultrafiltration as described above.Cloning of the purified PCR fragment into the linearized and purifiedpAILo2 vector was performed with an IN-FUSION™ Cloning Kit. The reaction(20 μl) contained 1× IN-FUSION™ Buffer, 1×BSA, 1 μl of IN-FUSION™ enzyme(diluted 1:10), 100 ng of pAILo2 digested with Nco I and Pac I, and 50ng of the Aspergillus fumigatus GH6A purified PCR product. The reactionwas incubated at room temperature for 30 minutes. A 2 μl sample of thereaction was used to transform transform E. coli TOP10 competent cellsaccording to the manufacturer's instructions. After the recovery period,two 100 μl aliquots from the transformation reaction were plated onto150 mm 2×YT plates supplemented with 100 μg of ampicillin per ml. Theplates were incubated overnight at 37° C. A set of eight putativerecombinant clones was selected at random from the selection plates andplasmid DNA was prepared from each one using a BIOROBOT® 9600. Cloneswere analyzed by Pst I restriction digest. Seven out of eight clones hadthe expected restriction digest pattern. Clones 1. 2 and 3 were thensequenced to confirm that there were no mutations in the cloned insert.Clone #2 was selected and designated pAILo33.

Aspergillus fumigatus cel6A (JaL355 ALLO33 Exp03191) was grown to obtainculture broth for the purification of a cellobiosehydrolase II.

Seven hundred and fifty ml of shake flask medium were added to a 2800 mlshake flask. The shake flask medium was composed per liter of 45 g ofmaltose, 2 g of K2HPO₄, 12 g of KH₂PO₄, 1 g of NaCl, 1 g of MgSO₄.7H₂O,7 g of yeast extract, 2 g of urea, and 0.5 ml of trace elementssolution. The trace elements solution was composed per liter of 13.8 gof FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, and 3 g of citric acid. Two shakeflasks were inoculated by suspension of a PDA plate of Aspergillusfumigatus cel6A with 0.01% TWEEN® 20 and incubated at 34° C. on anorbital shaker at 200 rpm for 120 hours.

The broth was filtered using 0.7 μm glass filter GF/F (Whatman,Piscataway, N.J., USA) and then using a 0.22 μm EXPRESS™ Plus Membrane(Millipore, Bedford, Mass., USA).

The filtered broth was concentrated and buffer exchanged using atangential flow concentrator (Pall Filtron, Northborough, Mass., USA)equipped with a 10 kDa polyethersulfone membrane (Pall Filtron,Northborough, Mass., USA) with 20 mM Tris-HCl pH 8.0. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 10: Preparation of Aspergillus terreus ATCC 28865 Cel7Endoglucanase I

The Aspergillus terreus ATCC 28865 Cel7 endoglucanase I gene (SEQ ID NO:19 [DNA sequence] and SEQ ID NO: 20 [deduced amino acid sequence]) wascloned and expressed in Aspergillus oryzae as described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the endoglucanase I gene from Aspergillus terreus ATCC 28865genomic DNA. Genomic DNA was isolated using a FASTDNA® Spin Kit for Soil(MP Biomedicals, Solon, Ohio, USA).

Primer #226: (SEQ ID NO: 103)5′-TAACAATTGTCACCATGAATTCTCTTACAAAAAGCAT-3′ Primer #227:(SEQ ID NO: 104) 5′-TATGCGGCCGCAGTCTGCATGTGTTACGCACCT-3′

The amplification reaction was composed of 1 μl of Aspergillus terreusATCC 28865 genomic DNA, 12.5 μl of 2× REDDYMIX™ PCR Buffer (ThermoFisher Scientific Inc., Waltham, Mass., USA), 1 μl of primer #226 (5μM), 1 μl of #227 (5 μM), and 9.5 μl of H2O. The amplification reactionwas incubated in a PTC-200 DNA ENGINE™ Thermal Cycler (MJ Research Inc.,Waltham, Mass., USA) programmed for 1 cycle at 94° C. for 2 minutes; and35 cycles each at 94° C. for 15 seconds and 60° C. for 1.5 minutes.

A 1.44 kb PCR reaction product was isolated by 1% agarose gelelectrophoresis using TAE buffer and staining with SYBR® Safe DNA gelstain (Invitrogen Corp., Carlsbad, Calif., USA). The DNA band wasvisualized with the aid of an EAGLE EYE® Imaging System (Stratagene, LaJolla, Calif., USA) and a DARKREADER® Transilluminator. The 1.44 kb DNAband was excised from the gel and purified using a GFX® PCR DNA and GelBand Purification Kit according to the manufacturer's instructions.

The 1.44 kb fragment was cleaved with Mfe I and Not I and purified usinga GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The cleaved 1.44 kb fragment was then directionally cloned by ligationinto Eco RI-Not I cleaved pXYG1051 (WO 2005/080559) using T4 ligase(Promega, Madison, Wis., USA) according to the manufacturer'sinstructions. The ligation mixture was transformed into E. coli-TOP10Fcompetent cells (Invitrogen Corp., Carlsbad, Calif., USA) according tothe manufacturer's instructions. The transformation mixture was platedonto LB plates supplemented with 100 μg of ampicillin per ml. Plasmidminipreps were prepared from several transformants and sequenced. Oneplasmid with the correct Aspergillus terreus GH7 coding sequence (SEQ IDNO: 13) was chosen. The plasmid was designated pXYG1051-NP003857. Theexpression vector pXYG1051 contains the same neutral amylase II (NA2)promoter derived from Aspergillus niger, and terminator elements aspCaHj483 (disclosed in Example 4 of WO 98/00529). Furthermore pXYG1051has pUC18 derived sequences for selection and propagation in E. coli,and pDSY82 (disclosed in Example 4 of U.S. Pat. No. 5,958,727) derivedsequences for selection and expression in Aspergillus facilitated by thepyrG gene of Aspergillus oryzae, which encodes orotidine decarboxylaseand is used to complement a pyrG mutant Aspergillus strain.

The expression plasmid pXYG1051-NP003857 was transformed intoAspergillus oryzae JaL355 as described in WO 98/00529. Transformantswere purified on selection plates through single conidia prior tosporulating them on PDA plates. Production of the Aspergillus terreusGH7 polypeptide by the transformants was analyzed from culturesupernatants of 1 ml 96 deep well stationary cultivations at 26° C. inYP medium with 2% maltodextrin. Expression was verified by SDS-PAGEusing a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM2-(N-morpholino)ethanesulfonic acid (MES) buffer (InvitrogenCorporation, Carlsbad, Calif., USA) by Coomassie blue staining. Onetransformant was selected for further work and designated Aspergillusoryzae 28.4.

For larger scale production, Aspergillus oryzae 28.4 spores were spreadonto a PDA plate and incubated for five days at 37° C. The confluentspore plate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspension was then used toinoculate twenty-five 500 ml flasks containing 100 ml of YPM medium. Theculture was incubated at 30° C. with constant shaking at 85 rpm. At dayfour post-inoculation, the culture broth was collected by filtrationthrough a triple layer of glass microfiber filters of 1.6 μm, 1.2 μm,and 0.7 μm (Whatman, Piscataway, N.J., USA). Fresh culture broth fromthis transformant produced a band of GH7 protein of approximately 64kDa. The identity of this band as the Aspergillus terreus GH7polypeptide was verified by peptide sequencing using standardtechniques.

Two liters of the filtered broth was concentrated to 400 ml and washedwith 50 mM HEPES pH 7.0 using a SARTOFLOW® Alpha Plus Crossflow Systemwith a 10 kDa cut-off (Sartorius Stedim Biotech S.A., Aubagne Cedex,France). Ammonium sulphate was added to a final concentration of 1 M anddissolved in the ultrafiltrate. The solution was loaded onto a Source 15Phenyl XK 26/20 50 ml column (GE Healthcare, HiHerod, Denmark). Afterloading the column was washed with 150 ml of 1 M ammonium sulphate andeluted with 1 column volume of 50% ethanol in a 0% to 100% gradientfollowed by 5 column volumes of 50% ethanol at a flow rate of 10 ml perminute. Fractions of 10 ml were collected and analyzed by SDS-PAGE.Fractions 3 to 8 were pooled and diluted to 1000 ml with 50 mM HEPES pH7.0 before loading onto a Q SEPHAROSE® Fast Flow XK26/20 60 ml column(GE Healthcare, HiHerod, Denmark). After loading the column was washed 3times with 60 ml of 50 mM HEPES pH 7.0 and eluded with 100 ml of 50 mMHEPES pH 7.0, 1 M NaCl at a flow rate of 10 ml per minute. Fractions of10 ml were collected and analyzed by SDS-PAGE. The flow through andfirst wash were pooled and concentrated to 400 ml and washed with 50 mMHEPES pH 7.0 using a SARTOFLOW® Alpha plus Crossflow System with a 10kDa cut-off. Further concentration was conducted using a VIVASPIN™centrifugal concentrator according to the manufacturer's instructions toa final volume of 80 ml. The protein concentration was determined byA₂₈₀/A₂₆₀ absorbance.

Example 11: Preparation of Trichoderma reesei RutC30 Cel5A EndoglucanaseII

The Trichoderma reesei RutC30 Cel5A endoglucanase II gene (SEQ ID NO: 21[DNA sequence] and SEQ ID NO: 22 [deduced amino acid sequence]) wascloned and expressed in Aspergillus oryzae as described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the endoglucanase II gene from Trichoderma reesei RutC30 genomicDNA. Genomic DNA was isolated using a DNEASY® Plant Maxi Kit. AnIN-FUSION™ PCR Cloning Kit was used to clone the fragment directly intopAILo2 (WO 2004/099228).

Forward primer: (SEQ ID NO: 105)5′-ACTGGATTTACCATGAACAAGTCCGTGGCTCCATTGCT-3′ Reverse primer:(SEQ ID NO: 106) 5′-TCACCTCTAGTTAATTAACTACTTTCTTGCGAGACACG-3′Bold letters represent coding sequence. The remaining sequence containssequence identity to insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 200 ng of Trichoderma reesei genomic DNA, 1×Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of 10 mM blend of dATP,dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA polymerase, and 1μl of 50 mM MgSO₄ in a final volume of 50 μl. The amplification reactionwas incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for onecycle at 98° C. for 2 minutes; and 35 cycles each at 94° C. for 30seconds, 61° C. for 30 seconds, and 68° C. for 1.5 minutes. After the 35cycles, the reaction was incubated at 68° C. for 10 minutes and thencooled at 10° C. A 1.5 kb PCR reaction product was isolated on a 0.8%GTG® agarose gel using TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of a DARKREADER™Transilluminator. The 1.5 kb DNA band was excised with a disposablerazor blade and purified using an ULTRAFREE® DA spin cup according tothe manufacturer's instructions.

Plasmid pAILo2 was linearized by digestion with Nco I and Pac I. Theplasmid fragment was purified by gel electrophoresis and ultrafiltrationas described above. Cloning of the purified PCR fragment into thelinearized and purified pAILo2 vector was performed using an IN-FUSION™PCR Cloning Kit. The reaction (20 μl) contained 1× IN-FUSION™ Buffer,1×BSA, 1 μl of IN-FUSION™ enzyme (diluted 1:10), 100 ng of pAILo2digested with Nco I and Pac I, and 100 ng of the Trichoderma reeseiCel5A endoglucanase II PCR product. The reaction was incubated at roomtemperature for 30 minutes. A 2 μl sample of the reaction was used totransform E. coli XL10 SOLOPACK® Gold cells according to themanufacturer's instructions. After a recovery period, two 100 μlaliquots from the transformation reaction were plated onto 150 mm 2×YTplates supplemented with 100 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. A set of 3 putative recombinant clones wasrecovered the selection plates and plasmid DNA was prepared from eachone using a BIOROBOT® 9600. Clones were analyzed by Pci I/Bsp LU11 Irestriction digestion. One clone with the expected restriction digestionpattern was then sequenced to confirm that there were no mutations inthe cloned insert. Clone #3 was selected and designated pAILo27.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Five micrograms of pAILo27(as well as pAILo2 as a control) were used to transform Aspergillusoryzae JaL250 protoplasts.

The transformation of Aspergillus oryzae JaL250 with pAILo27 yieldedabout 50 transformants. Eleven transformants were isolated to individualPDA plates and incubated for five days at 34° C.

Confluent spore plates were washed with 3 ml of 0.01% TWEEN® 80 and thespore suspension was used to inoculate 25 ml of MDU2BP medium in 125 mlglass shake flasks. Transformant cultures were incubated at 34° C. withconstant shaking at 200 rpm. At day five post-inoculation, cultures werecentrifuged at 6000×g and their supernatants collected. Five microlitersof each supernatant were mixed with an equal volume of 2× loading buffer(10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-glycineSDS-PAGE gel and stained with SIMPLYBLUE™ SafeStain (Invitrogen Corp.,Carlsbad, Calif., USA). SDS-PAGE profiles of the culture broths showedthat ten out of eleven transformants produced a new protein band ofapproximately 45 kDa. Transformant number 1, designated Aspergillusoryzae JaL250AILo27, was cultivated in a fermentor.

One hundred ml of shake flask medium were added to a 500 ml shake flask.The shake flask medium was composed per liter of 50 g of sucrose, 10 gof KH₂PO₄, 0.5 g of CaCl₂, 2 g of MgSO₄.7H₂O, 2 g of K₂SO₄, 2 g of urea,10 g of yeast extract, 2 g of citric acid, and 0.5 ml of trace metalssolution. The trace metals solution was composed per liter of 13.8 g ofFeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄. H₂O, 2.5 g ofCuSO₄.5H₂O, and 3 g of citric acid. The shake flask was inoculated withtwo plugs of Aspergillus oryzae JaL250AILo27 from a PDA plate andincubated at 34° C. on an orbital shaker at 200 rpm for 24 hours. Fiftyml of the shake flask broth was used to inoculate a 3 liter fermentationvessel.

A total of 1.8 liters of the fermentation batch medium was added to athree liter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). The fermentation batch medium was composed per liter of 10g of yeast extract, 24 g of sucrose, 5 g of (NH₄)₂SO₄, 2 g of KH₂PO₄,0.5 g of CaCl₂.2H₂O, 2 g of MgSO₄.7H₂O, 1 g of citric acid, 2 g ofK₂SO₄, 0.5 ml of anti-foam, and 0.5 ml of trace metals solution. Tracemetals solution was composed per liter of 13.8 g of FeSO₄.7H₂O, 14.3 gof ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g of CuSO₄.5H₂O, and 3 g ofcitric acid. Fermentation feed medium was composed of maltose.Fermentation feed medium was dosed at a rate of 0 to 4.4 g/l/hr for aperiod of 185 hours. The fermentation vessel was maintained at atemperature of 34° C. and pH was controlled using an Applikon 1030control system (Applikon Biotechnology, Schiedam, Netherlands) to aset-point of 6.1+/−0.1. Air was added to the vessel at a rate of 1 vvmand the broth was agitated by a Rushton impeller rotating at 1100 to1300 rpm. At the end of the fermentation, whole broth was harvested fromthe vessel and centrifuged at 3000×g to remove the biomass. Thesupernatant was sterile filtered and stored at 5 to 10° C.

The supernatant was desalted and buffer-exchanged in 20 mM Bis-Tris pH6.0 using a HIPREP® 26/10 desalting column according to themanufacturer's instructions. The buffer exchanged sample was loaded ontoa MonoQ® column (GE Healthcare, Piscataway, N.J., USA) equilibrated with20 mM Bis-Tris pH 6.0, and the bound protein was eluted with a lineargradient from 0 to 1000 mM sodium chloride. Protein fractions werepooled and buffer exchanged into 1.2 M (NH₄)₂SO₄-20 mM Tris-HCl pH 8.5.The sample was loaded onto a Phenyl SUPEROSE™ column (HR 16/10)equilibrated with 1.2 M (NH₄)₂SO₄−20 mM Tris-HCl pH 8.0. Bound proteinswere eluted with a linear gradient over 20 column volumes from 1.2 to 0M (NH₄)₂SO₄ in 20 mM Tris-HCl pH 8.5. The fractions were pooled,concentrated, and loaded onto a SUPERDEX® 75 HR 26/60 column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris-150 mMsodium chloride pH 8.5. Fractions were pooled and concentrated in 20 mMTris-150 mM sodium chloride pH 8.5. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 12: Preparation of Myceliophthora thermophila CBS 202.75 Cel5AEndoglucanase II

Myceliophthora thermophila CBS 202.75 Cel5A endoglucanase II (EGII) (SEQID NO: 23 [DNA sequence] and SEQ ID NO: 24 [deduced amino acidsequence]) was prepared recombinantly according to WO 2007/109441 usingAspergillus oryzae HowB104 as a host.

The culture filtrate was desalted and buffer-exchanged in 20 mM Tris pH8.0 using a HIPREP® 26/10 desalting column according to themanufacturer's instructions. The buffer exchanged sample was applied toa MonoQ® column equilibrated with 20 mM Tris pH 8.0, and the boundprotein was eluted with a gradient from 0 to 500 mM sodium chloride.Fractions were pooled and concentrated in 20 mM Tris pH 8.0. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 13: Preparation of Thermoascus aurantiacus CGMCC 0670 Cel5AEndoglucanase II

Thermoascus aurantiacus CGMCC 0670 cDNA encoding a Cel5A endoglucanaseII (SEQ ID NO: 25 [DNA sequence] and SEQ ID NO: 26 [deduced amino acidsequence]) was cloned according to the following procedure. The T.aurantiacus strain was grown in 80 ml of CBH1 medium (2.5% AVICEL®, 0.5%glucose, 0.14% (NH₄)₂SO₄) in 500 ml Erlenmeyer baffled flasks at 45° C.for 3 days with shaking at 165 rpm. Mycelia were harvested bycentrifugation at 7000 rpm for 30 minutes and stored at −80C before usefor RNA extraction. RNA was isolated from 100 mg of mycelia using aRNEASY® Plant Mini Kit.

The cDNA for the Thermoascus aurantiacus endoglucanase was isolated byRT PCR using a 3′ RACE system and a 5′ RACE system and primers BG025-1,BG025-2, BG025-3, and BG025-4 shown below to the N-terminal amino acids.

Primer BG025-1: (SEQ ID NO: 107)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC(T/C/A/G) GAATT-3′Primer BG025-2: (SEQ ID NO: 108)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC(T/C/A/G) GAGTT-3′Primer BG025-3: (SEQ ID NO: 109)5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G) GAATT-3′ Primer BG025-4:(SEQ ID NO: 110) 5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G) GAGTT-3′

The RT PCR products were ligated into plasmid pGEM-T using a pGEM-TVector System and transformed into E. coli strain JM109. A single cloneharboring a plasmid named pBGC1009 containing the endoglucanase cDNA wasisolated.

PCR primers were designed to amplify the cDNA encoding the T aurantiacusendoglucanase from plasmid pBGC1009. Restriction enzyme sites Bsp HI andPac I were incorporated for in-frame cloning into Aspergillus oryzaeexpression plasmid pBM120a (WO 2006/039541).

Primer 996261: (SEQ ID NO: 111) 5′-GATCTCATGAAGCTCGGCTCTCTCGT-3′ BspHIPrimer 996167: (SEQ ID NO: 112) 5′-TTAATTAATCAAAGATACGGAGTCAAAATAGG-3′PacI

The fragment of interest was amplified by PCR using an EXPAND™ HighFidelity PCR System. The PCR amplification reaction mixture contained 1μl of 0.09 μg/μl pBGC1009, 1 μl of primer 996261 (50 μmol/μl), 1 μl ofprimer 996167 (50 μmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μlof dNTP mix (10 mM each), 37.25 μl of water, and 0.75 μl (3.5 U/μl) ofDNA polymerase mix. An EPPENDORF® MASTERCYCLER® thermocycler was used toamplify the fragment programmed for 1 cycle at 94° C. for 2 minutes; 10cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds, 72° C. for1.5 minutes; 15 cycles each at 94° C. for 15 seconds, 55° C. for 30seconds, and 72° C. for 1.5 minutes plus 5 second elongation at eachsuccessive cycle; 1 cycle at 72° C. for 7 minutes; and a 4° C. hold.

The 1008 bp PCR product was purified by 1% agarose gel electrophoresisusing TAE buffer, excised from the gel, and purified using a QIAQUICK®Gel Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purifiedproduct was ligated directly into pCR®2.1-TOPO® according to themanufacturer's instructions. The resulting plasmid was named pBM124a.

Plasmid pBM124a was digested with Bsp HI and Pac 1, purified by 1%agarose gel electrophoresis using TAE buffer, excised from the gel, andpurified using a QIAQUICK® Gel Purification Kit. The plasmid fragmentwas ligated to the vector pBM120a, which was digested with Nco I and PacI. The resulting expression plasmid was designated pBM123a. PlasmidpBM123a contains a duplicate NA2-TPI promoter driving expression of theThermoascus aurantiacus endoglucanase cDNA clone, the AMG terminator,and amdS as a selectable marker.

Aspergillus oryzae BECh2 (WO 2000/139322) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra. Six μg ofpBM123a were used to transform Aspergillus oryzae BECh2. Primarytransformants were selected on COVE plates for 5 days. Transformantswere spore purified twice prior to shake flask analysis.

Spores of the transformants were inoculated into 25 ml of MY25 medium in125 ml shake flasks. The cultures were incubated at 34° C., 200 rpm on aplatform shaker for five days. On day 3 and day 5, culture supernatantswere harvested and clarified by centrifugation to remove mycelia. Twentymicroliters of supernatant from three transformants were analyzed usinga CRITERION® stain-free, 10-20% gradient SDS-PAGE gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. SDS-PAGE profiles of the cultures showedthat all transformants had a new major band of approximately 32 kDa. Onetransformant was chosen and named EXP00858.

Plastic, non-baffled 500 ml shake flasks containing 100 ml of SY50medium were inoculated with 0.1 ml of a spore stock of EXP00858, andincubated at 34° C., 200 rpm for 24 hours to produce a seed culture.Fifty ml of the seed culture was inoculated into a 2 liter fermentationtank containing 2 liters of medium composed per liter of 0.5 g ofpluronic acid, 30 g of sucrose, 2 g of MgSO₄.7H₂O, 2 g of anhydrousKH₂PO₄, 1 g of citric acid, 2 g of (NH₄)₂SO₄, 1 g of K₂SO₄, 20 g ofyeast extract, and 0.5 g of 200×AMG trace metals solution, pH 5.0. Thefermentation was fed with a maltose feed. The pH was controlled using 5NH₃PO₄ and 15% NH₄OH and maintained at 5.0 and then raised to 5.25.Temperature was maintained 34.0° C.+/−1.0° C. Agitation was 1000 rpm.Airflow was 1.0 vvm.

A 200 ml volume of cell-free supernatant was diluted to 1 liter withdeionized water. The pH was adjusted to 8 and the sample filtersterilized using a 0.22 μm polyethersulphone (PES) filter. The filtersterilized sample was loaded onto a 250 ml Q SEPHAROSE™ Fast Flow column(GE Healthcare, Piscataway, N.J., USA) pre-equilibrated with 25 mM TrispH 8. The enzyme was eluted from the column with a 0 to 1 M NaOHgradient in the same buffer. The fractions containing beta-glucosidaseactivity were pooled (400 ml) and the enzyme concentration calculatedfrom the theoretic extinction coefficient and the absorbance of thesample at 280 nm.

Example 14: Preparation of Aspergillus fumigatus NN055679 Cel3ABeta-Glucosidase

Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 27[DNA sequence] and SEQ ID NO: 28 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Trichodermareesei RutC30 as a host.

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator equipped with a 10 kDa polyethersulfone membrane with20 mM Tris-HCl pH 8.5. The sample was loaded onto a Q SEPHAROSE® HighPerformance column (GE Healthcare, Piscataway, N.J., USA) equilibratedin 20 mM Tris pH 8.5, and bound proteins were eluted with a lineargradient from 0-600 mM sodium chloride. The fractions were concentratedand loaded onto a SUPERDEX® 75 HR 26/60 column equilibrated with 20 mMTris-150 mM sodium chloride pH 8.5. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 15: Preparation of Penicillium brasilianum IBT 20888 Cel3ABeta-Glucosidase

Penicillium brasilianum IBT 20888 Cel3A beta-glucosidase (SEQ ID NO: 29[DNA sequence] and SEQ ID NO: 30 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2007/019442 using Aspergillusoryzae as a host.

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator equipped with a 10 kDa polyethersulfone membrane with20 mM Tris-HCl pH 8.0. The sample was loaded onto a Q SEPHAROSE® HighPerformance column (GE Healthcare, Piscataway, N.J., USA) equilibratedin 20 mM Tris pH 8.0, and bound proteins were eluted with a lineargradient from 0-600 mM sodium chloride. The fractions were concentratedinto 20 mM Tris pH 8.0. Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 16: Preparation of Aspergillus niger IBT 10140 Cel3Beta-Glucosidase

The Aspergillus niger IBT 10140 Cel3 beta-glucosidase gene (SEQ ID NO:31 [DNA sequence] and SEQ ID NO: 32 [deduced amino acid sequence]) wasisolated by PCR using two cloning primers GH3-9.1f and GH3-9.1r shownbelow, which were designed based on the publicly available AspergillusnigerCel3 sequence (CAK48740.1) for direct cloning using an IN-FUSION™Cloning Kit.

Primer GH3-9.1f: (SEQ ID NO: 113)acacaactggggatccaccatgaggttcacttcgatcgagg Primer GH3-9.1r:(SEQ ID NO: 114) agatctcgagaagcttaGTGAACAGTAGGCAGAGACGCCCG

A PCR reaction was performed with genomic DNA prepared from Aspergillusniger strain NN005810 in order to amplify the full-length gene. Thegenomic DNA was isolated using a FASTDNA® Spin Kit (MP Biomedicals,Santa Ana, Calif., USA). The PCR reaction was composed of 1 μl ofgenomic DNA, 0.75 μl of primer GH3-9.1f (10 μM), 0.75 μl of primerGH3-9.1r (10 μM), 3 μl of 5×HF buffer (Finnzymes Oy, Finland), 0.25 μlof 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μl of PHUSION® DNA polymerase(Finnzymes Oy, Finland), and PCR-grade water up to 15 μl. The PCRreaction was performed using a DYAD® PCR machine (Bio-Rad Laboratories,Inc., Hercules, Calif., USA) programmed for 2 minutes at 98° C. followedby 10 touchdown cycles at 98° C. for 15 seconds, 70° C. (−1° C./cycle)for 30 seconds, and 72° C. for 2 minutes 30 seconds; and 25 cycles eachat 98° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for 2 minutes 30seconds; and 5 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.6 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the A. niger Cel3 beta-glucosidase gene was cloned intothe expression vector pDAu109 (WO 2005/042735) linearized with Barn HIand Hind III, using an IN-FUSION™ Dry-Down PCR Cloning Kit according tothe manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kit(Omega Bio-Tek, Inc., Norcross, Ga., USA) according to themanufacturer's instructions. The A. niger Cel3 beta-glucosidase genesequence was verified by Sanger sequencing before heterologousexpression.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 10 mM CaCl₂-10 mM Tris-HCl pH 7.5-60% PEG 4000 (PEG4000; Applichem, Omaha, Nebr., USA) (polyethylene glycol, molecularweight 4,000) were added and gently mixed. The mixture was incubated at37° C. for 30 minutes and the protoplasts were spread on COVE plates fortransformant selection. After incubation for 4-7 days at 37° C., sporesof sixteen transformants were picked up and inoculated into YPM medium.After 4 days cultivation at 30° C. culture broth was analyzed in orderto identified the best transformants based on their ability to produceA. niger Cel3 beta-glucosidase. The screening was based on intensity ofthe band corresponding to the heterologous expressed protein determinedby SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-glucopyranoside (pNPG) using an assay was modifiedfrom Hagerdal et al., 1979, Biotechnology and Bioengineering 21:345-355: 10 μl of culture broth was mixed with 90 μl of assay reagentcontaining 10 μl of 0.1% TWEEN®, 10 μl of 1 M sodium citrate pH 5, 4 μlof 100 mM pNPG substrate (Sigma Aldrich) solubilized in DMSO (0.4% finalvolume in stock solution), and filtered water. The assay was incubatedfor 30 minutes at 37° C. and the absorbance was analyzed at 405 nmbefore and after addition of 100 μl of 1 M sodium carbonate pH 10. Thehighest absorbance values at 405 nm were correlated to the SDS-PAGE datafor selection of the best transformant.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then performed in 250 ml shake flasks using YP medium containing 2%maltodextrin for 4 days at 30° C. with shaking at 100 rpm.

A 2 liter volume of culture supernatant (EXP02895) was filtered with a0.7 μm glass fiber filter and then sterile filtered using a 0.22 μm PESmembrane (Nalgene Thermo Fisher Scientific, Rochester, N.Y., USA). Thefiltered supernatant was concentrated and diafiltered using cross-flowSartocon Slice Cassettes (non Cellulose) with 10 kDa cut-off (SartoriusStedim Biotech S.A., Aubagne Cedex, France). The final volume wasadjusted to 500 ml and pH adjusted to 4.5 by slowly adding dilute 10%acetic acid. The final ionic strength was under 4 MSi.

A 50 ml XK26 column (GE Healthcare, HiHerod, Denmark) was packed withXpressline ProA (UpFront Chromatography A/S, Copenhagen, Denmark)equilibrated with 50 mM sodium acetate pH 4.5 buffer. The filteredsupernatant was loaded onto the column using a P500 Pump (GE HealthCare, HiHerod, Denmark) at a flow of 45 ml per minute and washed with 50mM sodium acetate pH 4.5 buffer until all unbound material was eluted.The bound protein was eluted with 50 mM Tris pH 8 buffer using anÄKTAexplorer System (GE Healthcare, HiHerod, Denmark). Fractions werecollected and monitored by UV absorbance at 280 nm. The eluted proteinwere pooled and adjusted to pH 7 by slowly adding 0.5 M Tris base with afinal ionic strength under 4 MSi.

A 50 ml Q SEPHAROSE® Fast Flow column was equilibrated with 50 mM HEPESpH 7 buffer (buffer A). The column was then washed to remove unboundmaterial by washing with 50 mM HEPES pH 7 buffer until the UV absorbanceof the wash was below 0.05 at 280 nm. The bound protein was eluted usinga linear salt gradient of 0 to 1 M NaCl in 50 mM HEPES pH 7 buffer asbuffer B (10 column volume) using an ÄKTAexplorer System. Purity ofprotein fractions was determined by SDS-PAGE analysis using a 4-20%Tris-Glycine Gel (Invitrogen Carlsbad, Calif., USA) according to themanufacturer's instructions. Staining after electrophoresis wasperformed using INSTANT BLUE™ (Expedeon Ltd., Cambridgeshire, UK)according to the manufacturer's instructions. Fractions showing expectedprotein bands were pooled. Identification of the protein was determinedby MS-Edman degradation using standard methods.

Example 17: Preparation of Thermoascus aurantiacus CGMCC 0583 GH61APolypeptide Having Cellulolytic Enhancing Activity

Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide having cellulolyticenhancing activity (SEQ ID NO: 33 [DNA sequence] and SEQ ID NO: 34[deduced amino acid sequence]) was recombinantly prepared according toWO 2005/074656 using Aspergillus oryzae JaL250 as a host. Therecombinantly produced Thermoascus aurantiacus GH61A polypeptide wasfirst concentrated by ultrafiltration using a 10 kDa membrane, bufferexchanged into 20 mM Tris-HCl pH 8.0, and then purified using a 100 ml QSEPHAROSE® Big Beads column (GE Healthcare, Piscataway, N.J., USA) with600 ml of a 0-600 mM NaCl linear gradient in the same buffer. Fractionsof 10 ml were collected and pooled based on SDS-PAGE.

The pooled fractions (90 ml) were then further purified using a 20 mlMONO Q® column (GE Healthcare, Piscataway, N.J., USA) with 500 ml of a0-500 mM NaCl linear gradient in the same buffer. Fractions of 6 ml werecollected and pooled based on SDS-PAGE. The pooled fractions (24 ml)were concentrated by ultrafiltration using a 10 kDa membrane, andchromatographed using a 320 ml SUPERDEX® 75 SEC column (GE Healthcare,Piscataway, N.J., USA) with isocratic elution of approximately 1.3liters of 150 mM NaCl-20 mM Tris-HCl pH 8.0. Fractions of 20 ml werecollected and pooled based on SDS-PAGE. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 18: Preparation of Thielavia terrestris NRRL 8126 GH61EPolypeptide Having Cellulolytic Enhancing Activity

Thielavia terrestris NRRL 8126 GH61E polypeptide having cellulolyticenhancing activity (SEQ ID NO: 35 [DNA sequence] and SEQ ID NO: 36[deduced amino acid sequence]) was recombinantly prepared according toU.S. Pat. No. 7,361,495 using Aspergillus oryzae JaL250 as a host.

Filtered culture broth was desalted and buffer-exchanged into 20 mMsodium acetate-150 mM NaCl pH 5.0 using a HIPREP® 26/10 Desalting Columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 19: Preparation of Aspergillus fumigatus NN051616 GH61BPolypeptide Having Cellulolytic Enhancing Activity

A tblastn search (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402) of the Aspergillus fumigatus partial genome sequence (TheInstitute for Genomic Research, Rockville, Md.) was performed using asquery several known GH61 proteins including GH61A from Thermoascusaurantiacus (GeneSeqP Accession Number AEC05922). Several genes wereidentified as putative Family GH61 homologs based upon a high degree ofsimilarity to the query sequences at the amino acid level. One genomicregion of approximately 850 bp with greater than 70% identity to theThermoascus aurantiacus GH61A sequence at the amino acid level waschosen for further study.

Aspergillus fumigatus NN051616 was grown and harvested as described inU.S. Pat. No. 7,244,605. Frozen mycelia were ground, by mortar andpestle, to a fine powder and genomic DNA was isolated using a DNEASY®Plant Kit according to manufacturer's instructions.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Aspergillus fumigatus Family GH61B protein gene from thegenomic DNA. An IN-FUSION® Cloning Kit was used to clone the fragmentdirectly into the expression vector pAILo2, without the need forrestriction digestion and ligation.

Forward primer: (SEQ ID NO: 115)5′-ACTGGATTTACCATGACTTTGTCCAAGATCACTTCCA-3′ Reverse primer:(SEQ ID NO: 116) 5′-TCACCTCTAGTTAATTAAGCGTTGAACAGTGCAGGACCAG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 204 ng of Aspergillus fumigatus genomic DNA, 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 1.5 μl of 10mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNAPolymerase, and 1 μl of 50 mM MgSO₄ in a final volume of 50 μl. Theamplification was performed using an EPPENDORF® MASTERCYCLER® 5333epgradient S (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for one cycle at 94° C. for 3 minutes; and 30 cycles each at94° C. for 30 seconds, 56° C. for 30 seconds, and 72° C. for 1 minutes.The heat block was then held at 72° C. for 15 minutes followed by a 4°C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 850 bp product band was excisedfrom the gel and purified using a MINELUTE® Gel Extraction Kit (QIAGENInc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The fragment was then cloned into pAILo2 using an IN-FUSION® CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis as above and a QIAQUICK® GelPurification Kit. The gene fragment and the digested vector werecombined together in a reaction resulting in the expression plasmidpAG43 in which transcription of the Family GH61B protein gene was underthe control of the NA2-tpi promoter. The recombination reaction (20 μl)was composed of 1× IN-FUSION® Buffer, 1×BSA, 1 μl of IN-FUSION® enzyme(diluted 1:10), 166 ng of pAILo2 digested with Nco I and Pac I, and 110ng of the Aspergillus fumigatus GH61B protein purified PCR product. Thereaction was incubated at 37° C. for 15 minutes followed by 15 minutesat 50° C. The reaction was diluted with 40 μl of 10 mM Tris-0.1 M EDTAbuffer and 2.5 μl of the diluted reaction was used to transform E. coliSOLOPACK® Gold cells. An E. coli transformant containing pAG43 (GH61Bprotein gene) was identified by restriction enzyme digestion and plasmidDNA was prepared using a BIOROBOT® 9600.

DNA sequencing of the 862 bp PCR fragment was performed with aPerkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencerusing dye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. The following vector specific primers were used forsequencing:

pAllo2 5 Seq: (SEQ ID NO: 117) 5′ TGTCCCTTGTCGATGCG 3′ pAllo2 3 Seq:(SEQ ID NO: 118) 5′ CACATGACTTGGCTTCC 3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

A gene model for the Aspergillus fumigatus sequence was constructedbased on similarity of the encoded protein to the Thermoascusaurantiacus GH61A protein (GeneSeqP Accession Number AEC05922). Thenucleotide sequence and deduced amino acid sequence, are shown in SEQ IDNO: 37 and SEQ ID NO: 38, respectively. The genomic fragment encodes apolypeptide of 250 amino acids, interrupted by 2 introns of 53 and 56bp. The % G+C content of the gene and the mature coding sequence are53.9% and 57%, respectively. Using the SignalP software program (Nielsenet al., 1997, Protein Engineering 10: 1-6), a signal peptide of 21residues was predicted. The predicted mature protein contains 221 aminoacids with a predicted molecular mass of 23.39 kDa.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Six μg of pAG43 were used totransform Aspergillus oryzae JaL355. Twenty-six transformants wereisolated to individual PDA plates.

Confluent PDA plates of 24 transformants were each washed with 5 ml of0.01% TWEEN® 20 and the spores were each collected. Eight μl of eachspore stock was added to 1 ml of YPG, YPM, and M410 media separately in24 well plates and incubated at 34° C. After 3 days of incubation, 7.5μl of supernatant from four transformants were analyzed using aCRITERION® stain-free, 8-16% gradient SDS-PAGE gel according to themanufacturer's instructions. Based on this gel, M410 was chosen as thebest medium. Five days after incubation, 7.5 μl of supernatant from eachM410 culture was analyzed using a CRITERION® stain-free, 8-16% gradientSDS-PAGE gel. SDS-PAGE profiles of the cultures showed that severaltransformants had a new major band of approximately 25 kDa.

A confluent plate of one transformant (grown on PDA) was washed with 5ml of 0.01% TWEEN® 20 and inoculated into four 500 ml Erlenmeyer flaskscontaining 100 ml of M410 medium to generate broth for characterizationof the enzyme. The flasks were harvested on day 5 (300 ml), filteredusing a 0.22 μm EXPRESS™ Plus Membrane, and stored at 4° C.

The filtered shake flask broth containing Aspergillus fumigatus GH61Bpolypeptide having cellulolytic enhancing activity was concentratedusing a 10 kDa MWCO Amicon Ultra centrifuge concentrator (Millipore,Bedford, Mass., USA) to approximately 10-fold smaller volume. Theconcentrated filtrate was buffer-exchanged and desalted using a BIO-GEL®P6 desalting column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA)pre-equilibrated in 20 mM Tris-(hydroxymethyl)aminomethane (Sigma, St.Louis, Mo., USA), pH 8.0, according to the manufacturer's instructionswith the following exception: 3 ml of sample was loaded and eluted with3 ml of buffer. Concentrated, desalted GH61B protein was quantifiedusing a BCA protein assay using bovine serum albumin as a proteinconcentration standard. Quantification was performed in triplicate.Enzyme purity was confirmed using 8-16% gradient SDS-PAGE at 200 voltsfor 1 hour and staining with BIO-SAFE™ Coomassie Stain.

Example 20: Preparation of Penicillium pinophilum GH61 PolypeptideHaving Cellulolytic Enhancing Activity

Penicillium pinophilum GH61 polypeptide having cellulolytic enhancingactivity SEQ ID NO: 39 [DNA sequence] and SEQ ID NO: 40 [deduced aminoacid sequence]) was preaped according to the procedure described below.

Compost samples were collected from Yunnan, China on Dec. 12, 2000.Penicillium pinophilum NN046877 was isolated using single sporeisolation techniques on PDA plates at 45° C. Penicillium pinophilumstrain NN046877 was inoculated onto a PDA plate and incubated for 4 daysat 37° C. in the darkness. Several mycelia-PDA plugs were inoculatedinto 500 ml shake flasks containing 100 ml of NNCYP-PCS medium. Theflasks were incubated for 5 days at 37° C. with shaking at 160 rpm. Themycelia were collected at day 4 and day 5. The mycelia from each daywere frozen in liquid nitrogen and stored in a −80° C. freezer untiluse.

The frozen mycelia were transferred into a liquid nitrogen prechilledmortar and pestle and ground to a fine powder. Total RNA was preparedfrom the powdered mycelia of each day by extraction with TRIZOL™ reagent(Invitrogen Corporation, Carlsbad, Calif., USA). The polyA enriched RNAwas isolated using a mTRAP™ Total Kit (Active Motif, Carlsbad, Calif.,USA).

Double stranded cDNA from each day was synthesized with a SMART™ cDNAlibrary Construction Kit (Clontech Laboratories, Inc., Mountain View,Calif., USA). The cDNA was cleaved with Sfi I and the cDNA was sizefractionated by 0.8% agarose gel electrophoresis using TBE buffer. Thefraction of cDNA of 500 bp and larger was excised from the gel andpurified using a GFX® PCR DNA and Gel Band Purification Kit according tothe manufacturer's instructions. Then equal amounts of cDNA from day 4and day 5 were pooled for library construction.

The pooled cDNA was then directionally cloned by ligation into Sfi Icleaved pMHas7 (WO 2009/037253) using T4 ligase (New England Biolabs,Inc., Beverly, Mass., USA) according to the manufacturer's instructions.The ligation mixture was electroporated into E. coli ELECTROMAX™ DH10B™cells (Invitrogen Corp., Carlsbad, Calif., USA) using a GENE PULSER® andPulse Controller (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at25 uF, 25 mAmp, 1.8 kV with a 1 mm gap cuvette according to themanufacturer's procedure.

The electroporated cells were plated onto LB plates supplemented with 50mg of kanamycin per liter. A cDNA plasmid pool was prepared from 60,000total transformants of the original pMHas7 vector ligation. Plasmid DNAwas prepared directly from the pool of colonies using a QIAGEN® PlasmidKit (QIAGEN Inc., Valencia, Calif., USA).

A transposon containing plasmid designated pSigA4 was constructed fromthe pSigA2 transposon containing plasmid described WO 2001/77315 inorder to create an improved version of the signal trapping transposon ofpSigA2 with decreased selection background. The pSigA2 transposoncontains a signal less beta-lactamase construct encoded on thetransposon itself. PCR was used to create a deletion of the intact betalactamase gene found on the plasmid backbone using a proofreading PfuTurbo polymerase PROOFSTART™ (QIAGEN GmbH Corporation, Hilden, Germany)and the following 5′ phosphorylated primers (TAG Copenhagen, Denmark):

SigA2NotU-P: (SEQ ID NO: 119) 5′-TCGCGATCCGTTTTCGCATTTATCGTGAAACGCT-3′SigA2NotD-P: (SEQ ID NO: 120) 5′-CCGCAAACGCTGGTGAAAGTAAAAGATGCTGAA-3′

The amplification reaction was composed of 1 μl of pSigA2 (10 ng/μl), 5μl of 10× PROOFSTART™ Buffer (QIAGEN GmbH Corporation, Hilden, Germany),2.5 μl of dNTP mix (20 mM), 0.5 μl of SigA2NotU-P (10 mM), 0.5 μl ofSigA2NotD-P (10 mM), 10 μl of Q solution (QIAGEN GmbH Corporation,Hilden, Germany), and 31.25 μl of deionized water. A DNA ENGINE™ ThermalCycler (MJ Research Inc., Waltham, Mass., USA) was used foramplification programmed for one cycle at 95° C. for 5 minutes; and 20cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for 4 minutes.

A 3.9 kb PCR reaction product was isolated by 0.8% agarose gelelectrophoresis using TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of an EAGLE EYE® Imaging System(Stratagene, La Jolla, Calif., USA) at 360 nm. The 3.9 kb DNA band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 3.9 kb fragment was self-ligated at 16° C. overnight with 10 unitsof T4 DNA ligase (New England Biolabs, Inc., Beverly, Mass., USA), 9 μlof the 3.9 kb PCR fragment, and 1 μl of 10× ligation buffer (New EnglandBiolabs, Inc., Beverly, Mass., USA). The ligation was heat inactivatedfor 10 minutes at 65° C. and then digested with Dpn I at 37° C. for 2hours. After incubation, the digestion was purified using a GFX® PCR DNAand Gel Band Purification Kit.

The purified material was then transformed into E. coli TOP10 competentcells according to the manufacturer's instructions. The transformationmixture was plated onto LB plates supplemented with 25 μg ofchloramphenicol per ml. Plasmid minipreps were prepared from severaltransformants and digested with Bgl II. One plasmid with the correctconstruction was chosen. The plasmid was designated pSigA4. PlasmidpSigA4 contains the Bgl II flanked transposon SigA2 identical to thatdisclosed in WO 2001/77315.

A 60 μl sample of plasmid pSigA4 DNA (0.3 μg/μl) was digested with BglII and separated by 0.8% agarose gel electrophoresis using TBE buffer. ASigA2 transposon DNA band of 2 kb was eluted with 200 μl of EB buffer(QIAGEN GmbH Corporation, Hilden, Germany) and purified using a GFX® PCRDNA and Gel Band Purification Kit according to the manufacturer'sinstructions and eluted in 200 μl of EB buffer. SigA2 was used fortransposon assisted signal trapping.

A complete description of transposon assisted signal trapping isdescribed in WO 2001/77315. The plasmid pool was treated with transposonSigA2 and HYPERMU™ transposase (EPICENTRE Biotechnologies, Madison,Wis., USA) according to the manufacturer's instructions.

For in vitro transposon tagging of the Penicillium pinophilum cDNAlibrary, 2 μl of SigA2 transposon containing approximately 100 ng of DNAwere mixed with 1 μl of the plasmid DNA pool of the Penicilliumpinophilum cDNA library containing 1 μg of DNA, 1 μl of HYPERMU™transposase, and 2 μl of 10× buffer (EPICENTRE Biotechnologies, Madison,Wis., USA) in a total volume of 20 μl and incubated at 30° C. for 3hours followed by adding 2 μl of stop buffer (EPICENTRE Biotechnologies,Madison, Wis., USA) and heat inactivation at 75° C. for 10 minutes. TheDNA was precipitated by addition of 2 μl of 3 M sodium acetate pH 5 and55 μl of 96% ethanol and centrifuged for 30 minutes at 10,000×g, 4° C.The pellet was washed in 70% ethanol, air dried at room temperature, andresuspended in 10 μl of deionized water.

A 2 μl volume of the transposon tagged plasmid pool was electroporatedinto 50 μl of E. coli ELECTROMAX™ DH10B™ cells (Invitrogen Corp.,Carlsbad, Calif., USA) according to the manufacturer's instructionsusing a GENE PULSER® and Pulse Controller at 25 uF, 25 mAmp, 1.8 kV witha 1 mm gap cuvette according to the manufacturer's procedure.

The electroporated cells were incubated in SOC medium with shaking at225 rpm for 1 hour at 37° C. before being plated onto the followingselective media: LB medium supplemented with 50 μg of kanamycin per ml;LB medium supplemented with 50 μg of kanamycin per ml and 15 μg ofchloramphencol per ml; and LB medium supplemented with 50 μg ofkanamycin per ml, 15 μg of chloramphencol per ml, and 30 μg ofampicillin per ml.

From plating of the electroporation onto LB medium supplemented withkanamycin, chloramphencol and ampicillin, approximately 200 colonies per50 μl were observed after 3 days at 30° C. All colonies were replicaplated onto LB kanamycin, chloramphenicol, and ampicillin mediumdescribed above. Five hundred colonies were recovered under thisselection condition. The DNA from each colony was sequenced with thetransposon forward and reverse primers (primers A and B), shown below,according to the procedure disclosed in WO 2001/77315 (page 28).

Primer A: (SEQ ID NO: 121) 5′-agcgtttgcggccgcgatcc-3′ Primer B:(SEQ ID NO: 122) 5′-ttattcggtcgaaaaggatcc-3′

DNA sequences were obtained from SinoGenoMax Co., Ltd (Beijing, China).Primer A and primer B sequence reads for each plasmid were trimmed toremove vector and transposon sequence. The assembled sequences weregrouped into contigs by using the program PhredPhrap (Ewing et al.,1998, Genome Research 8: 175-185; Ewing and Green, 1998, Genome Research8: 186-194). All contigs were subsequently compared to sequencesavailable in standard public DNA and protein sequences databases(TrEMBL, SWALL, PDB, EnsemblPep, GeneSeqP) using the program BLASTX2.0a19MP-WashU [14 Jul. 1998] [Build linux-x86 18:51:44 30 Jul. 1998](Gish et al., 1993, Nat. Genet. 3: 266-72). The family GH10 xylanasecandidate was identified directly by analysis of the BlastX results.

Penicillium pinophilum NN046877 was grown on a PDA agar plate at 37° C.for 4-5 days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Based on the Penicillium pinophilum GH10 xylanase gene informationobtained as described above, oligonucleotide primers, shown below, weredesigned to amplify the GH61 gene from genomic DNA of Penicilliumpinophilum GH10 NN046877. An IN-FUSION® CF Dry-down Cloning Kit was usedto clone the fragment directly into the expression vector pPFJO355,without the need for restriction digestion and ligation.

Sense primer: SEQ ID NO: 123)5′-ACACAACTGGGGATCCACCATGACTCTAGTAAAGGCTATTCT TTTAGC-3′Antisense primer: (SEQ ID NO: 124)5′-GTCACCCTCTAGATCTTCACAAACATTGGGAGTAGTATGG-3′Bold letters represented the coding sequence and the remaining sequencewas homologous to insertion sites of pPFJO355.

The expression vector pPFJO355 contains the Aspergillus oryzaeTAKA-amylase promoter, Aspergillus niger glucoamylase terminatorelements, pUC19 derived sequences for selection and propagation in E.coli, and an Aspergillus nidulans pyrG gene, which encodes an orotidinedecarboxylase for selection of a transformant of a pyrG mutantAspergillus strain.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium sp. NN051602 genomic DNA, 10 μl of 5×GCBuffer (Finnzymes Oy, Espoo, Finland), 1.5 μl of DMSO, 2.5 mM each ofdATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ High-Fidelity DNAPolymerase (Finnzymes Oy, Espoo, Finland), in a final volume of 50 μl.The amplification was performed using a Peltier Thermal Cycler (MJResearch Inc., South San Francisco, Calif., USA) programmed fordenaturing at 98° C. for 1 minutes; 5 cycles of denaturing at 98° C. for15 seconds, annealing at 56° C. for 30 seconds, with a 1° C. increaseper cycle and elongation at 72° C. for 75 seconds; and 25 cycles each at98° C. for 15 seconds, 65C for 30 seconds and 72° C. for 75 seconds; anda final extension at 72° C. for 10 minutes. The heat block then went toa 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.4 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION® CF Dry-down PCR Cloning Kit resulting in pPpin3 in whichtranscription of the Penicillium pinophilum GH10 xylanase gene was underthe control of a promoter from the gene for Aspergillus oryzaealpha-amylase. In brief, 30 ng of pPFJO355 digested with Barn I and BglII, and 60 ng of the Penicillium pinophilum GH10 xylanase gene purifiedPCR product were added to a reaction vial and resuspended in a finalvolume of 10 μl with addition of deionized water. The reaction wasincubated at 37° C. for 15 minutes and then 50° C. for 15 minutes. Threeμl of the reaction were used to transform E. coli TOP10 competent cells.An E. coli transformant containing pPpin3 was detected by colony PCR andplasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc.,Valencia, Calif., USA). The Penicillium pinophilum GH10 xylanase geneinsert in pPpin3 was confirmed by DNA sequencing using a 3730XL DNAAnalyzer (Applied Biosystems Inc, Foster City, Calif., USA).

The same PCR fragment was cloned into vector pGEM-T using a pGEM-TVector System to generate pGEM-T-Ppin3. The Penicillium pinophilum GH10xylanase gene contained in pGEM-T-Ppin3 was confirmed by DNA sequencingusing a 3730XL DNA Analyzer. E. coli strain T-Ppin3, containingpGEM-T-Ppin3, was deposited with the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSM), D-38124 Braunschweig,Germany on Sep. 7, 2009, and assigned accession number DSM 22922.

DNA sequencing of the Penicillium pinophilum genomic clone encoding aGH10 polypeptide having xylanase activity was performed with an AppliedBiosystems Model 3700 Automated DNA Sequencer using version 3.1 BIG-DYE™terminator chemistry (Applied Biosystems, Inc., Foster City, Calif.,USA) and dGTP chemistry (Applied Biosystems, Inc., Foster City, Calif.,USA) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of thePenicillium pinophilum gh10 gene are shown in SEQ ID NO: 39 and SEQ IDNO: 40, respectively. The coding sequence is 1442 bp including the stopcodon and is interrupted by three introns of 51 bp (199-249), 73 bp(383-455), and 94 bp (570-663). The encoded predicted protein is 407amino acids. The % G+C of the coding sequence of the gene (includingintrons) is 47.99% G+C and the mature polypeptide coding sequence is49.22%. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 388 amino acids with a predictedmolecular mass of 41.5 kDa and an isoelectric pH of 5.03.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Penicillium pinophilum gene encoding the GH10polypeptide having xylanase activity shares 76% and 87% identity(excluding gaps) to the deduced amino acid sequence of a predicted GH10family protein from Talaromyces emersonii (AAU99346) and Penicilliummarneffei (B6QN64), respectively.

Aspergillus oryzae HowB101 (WO 95/35385 Example 1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Three μg of pPpin3 were transformed intoAspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pPpin3 yieldedabout 50 transformants. Twelve transformants were isolated to individualMinimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C. with shaking at 150 rpm. After 3days incubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT® Blue (Expedeon Ltd., Babraham Cambridge, UK).SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 55 kDa. The expressionstrain was designated Aspergillus oryzae EXP02765.

A slant of one transformant, designated transformant 2, was washed with10 ml of YPM and inoculated into a 2 liter flask containing 400 ml ofYPM medium to generate broth for characterization of the enzyme. Theculture was harvested on day 3 and filtered using a 0.45 μm DURAPORE®Membrane (Millipore, Bedford, Mass., USA).

A 1 liter volume of supernatant of the recombinant Aspergillus oryzaestrain EXP02765 was precipitated with ammonium sulfate (80% saturation)and redissolved in 50 ml of 25 mM sodium acetate pH 4.3, and thendialyzed against the same buffer and filtered through a 0.45 μm filter.The solution was applied to a 40 ml Q SEPHAROSE™ Fast Flow column columnequilibrated in 25 mM sodium acetate pH 4.3. The recombinant GH10protein did not bind to the column. The fractions with xylanase activitywere collected and evaluated by SDS-PAGE as described above. Fractionscontaining a band of approximately 55 kDa were pooled. The pooledsolution was concentrated by ultrafiltration.

Example 21: Preparation of Penicillium sp. GH61 Polypeptide HavingCellulolytic Enhancing Activity

Penicillium sp. GH61 polypeptide having cellulolytic enhancing activitySEQ ID NO: 41 [DNA sequence] and SEQ ID NO: 42 [deduced amino acidsequence]) according to the following procedure.

A compost sample was collected from Yunnan, China. Penicillium sp.NN051602 was isolated using single spore isolation techniques on PDAplates at 45° C. The Penicillium sp. strain was inoculated onto a PDAplate and incubated for 4 days at 45° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of NNCYP-PCS medium. The flasks were incubated for 6 days at 45°C. with shaking at 160 rpm. The mycelia were collected at day 4, day 5,and day 6. Then the mycelia from each day were combined and frozen inliquid nitrogen, and then stored in a −80° C. freezer until use.

The frozen mycelia were transferred into a liquid nitrogen prechilledmortar and pestle and ground to a fine powder. Total RNA was preparedfrom the powdered mycelia by extraction with TRIZOL® reagent andpurified using a RNEASY® Mini Kit according to the manufacturer'sprotocol. Fifty micrograms of total RNA was submitted to sequencing asdescribed above.

Total RNA enriched for polyA sequences with the mRNASeq protocol wassequenced using an ILLUMINA® GA2 system (Illumina, Inc., San Diego,Calif., USA). The raw 36 base pair reads were assembled with an in-housedeveloped assembler. The assembled sequences were analyzed usingstandard bioinformatics methods for gene finding and functionalprediction. ESTscan 2.0 was used for gene prediction. NCBI blastaIIversion 2.2.10 and HMMER version 2.1.1 were used to predict functionbased on structural homology. The Family GH61 candidate was identifieddirectly by analysis of the Blast results.

Penicillium sp. NN051602 was grown on a PDA agar plate at 45° C. for 3days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Based on the ILLUMINA® sequencing information of the Penicillium sp.GH61 gene obtained as described above, oligonucleotide primers, shownbelow, were designed to amplify the GH61 gene from genomic DNA ofPenicillium sp. NN051602. An IN-FUSION® CF Dry-down Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) was used toclone the fragment directly into the expression vector pPFJO355, withoutthe need for restriction digestion and ligation.

Sense primer: (SEQ ID NO: 125)5′-ACACAACTGGGGATCCACCATGCTGTCTTCGACGACTCGCA-3′ Antisense primer:(SEQ ID NO: 126) 5′-GTCACCCTCTAGATCTCGACTTCTTCTAGAACGTCGGCTCA-3′Bold letters represented the coding sequence and the remaining sequencewas homologous to insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium sp. NN051602 genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and0.6 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo,Finland) in a final volume of 50 μl. The amplification was performedusing a Peltier Thermal Cycler programmed for denaturing at 98° C. for 1minutes; 5 cycles of denaturing at 98° C. for 15 seconds, annealing at63° C. for 30 seconds, with a 1° C. increase per cycle and elongation at72° C. for 60 seconds; and 25 cycles each at 98° C. for 15 seconds and72° C. for 60 seconds; and a final extension at 72° C. for 5 minutes.The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 0.9 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION® CF Dry-down PCR Cloning Kit resulting in pGH61D23Y4 in whichtranscription of the Penicillium sp. GH61 gene was under the control ofa promoter from the gene for Aspergillus oryzae alpha-amylase. In brief,30 ng of pPFJO355 digested with Barn I and Bgl II, and 60 ng of thePenicillium sp. GH61 gene purified PCR product were added to a reactionvial and resuspended in a final volume of 10 μl with addition ofdeionized water. The reaction was incubated at 37° C. for 15 minutes andthen 50° C. for 15 minutes. Three μl of the reaction were used totransform E. coli TOP10 competent cells. An E. coli transformantcontaining pGH61D23Y4 was detected by colony PCR and plasmid DNA wasprepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). The Penicillium sp. GH61 gene insert in pGH61D23Y4 wasconfirmed by DNA sequencing using a 3730XL DNA Analyzer.

The same PCR fragment was cloned into vector pGEM-T using a pGEM-TVector System to generate pGEM-T-GH61D23Y4. The Penicillium sp. GH61gene insert in pGEM-T-GH61D23Y4 was confirmed by DNA sequencing using a3730XL DNA Analyzer. E. coli strain T-51602, containingpGEM-T-GH61D23Y4, was deposited with the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSM), Mascheroder Weg 1 B,D-38124 Braunschweig, Germany on Aug. 26, 2009 and assigned accessionnumber DSM 22882.

DNA sequencing of the Penicillium sp. genomic clone encoding a GH61Apolypeptide having cellulolytic-enhancing activity was performed with anApplied Biosystems Model 3700 Automated DNA Sequencer using version 3.1BIG-DYE™ terminator chemistry (Applied Biosystems, Inc., Foster City,Calif., USA) and dGTP chemistry (Applied Biosystems, Inc., Foster City,Calif., USA) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of thePenicillium sp. gh61a gene are shown in SEQ ID NO: 45 and SEQ ID NO: 46,respectvely. The coding sequence is 835 bp including the stop codon andis interrupted by one intron of 73 bp (114-186). The encoded predictedprotein is 253 amino acids. The % G+C of the coding sequence of the gene(including introns) is 63.35% G+C and the mature polypeptide codingsequence is 64.62%. Using the SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6), a signal peptide of 25 residues waspredicted. The predicted mature protein contains 228 amino acids with apredicted molecular mass of 24.33 kDa and an isoelectric pH of 4.17.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Penicillium gene encoding the GH61A polypeptide havingcellulolytic-enhancing activity shares 74% identity (excluding gaps) tothe deduced amino acid sequence of a predicted GH61 family protein fromThermoascus aurantiacus (GENESEQP:AUM 17198).

Aspergillus oryzae HowB101 (WO 95/35385 Example 1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Three μg of pGH61D23Y4 were used totransform Aspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pGH61D23Y4 yieldedabout 50 transformants. Twelve transformants were isolated to individualMinimal medium plates.

Six transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed on aNUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer according to themanufacturer's instructions. The resulting gel was stained with INSTANT®Blue (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profiles of thecultures showed that the majority of the transformants had a major bandof approximately 45 kDa. The expression strain was designated asAspergillus oryzae EXP03089.

A slant of one transformant, designated transformant 1, was washed with10 ml of YPM medium and inoculated into a 2 liter flask containing 400ml of YPM medium to generate broth for characterization of the enzyme.The culture was harvested on day 3 and filtered using a 0.45 μm DURAPOREMembrane (Millipore, Bedford, Mass., USA).

A 400 ml volume of the filtered broth of the recombinant strainAspergillus oryzae EXP03089 was precipitated with ammonium sulfate (80%saturation) and redissolved in 20 ml of 25 mM sodium acetate pH 5.0buffer, and then dialyzed against the same buffer and filtered through a0.45 μm filter. The solution was applied to a 30 ml Q SEPHAROSE® FastFlow column (GE Healthcare, Buckinghamshire, UK) equilibrated in 25 mMsodium acetate pH 5.0. The recombinant GH61 protein was eluted with alinear NaCl gradient (0-0.4 M). Fractions eluted with 0.1-0.2 M NaClwere collected and dialyzed against the same equilibration buffer. Thesample was further purified on a MONO Q® column (GE Healthcare,Buckinghamshire, UK) with a linear NaCl gradient (0-0.3 M). Fractionswere evaluated by SDS-PAGE. Fractions containing a band of approximately45 kDa were pooled. The pooled solution was concentrated byultrafiltration.

Example 22: Preparation of Thielavia terrestris GH61N Polypeptide HavingCellulolytic Enhancing Activity

Thielavia terrestris NRRL 8126 (SEQ ID NO: 43 [DNA sequence] and SEQ IDNO: 44 [deduced amino acid sequence]) was prepared according to thefollowing procedure.

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). A preliminary assembly of thegenome was downloaded from JGI and analyzed using the Pedant-Pro™Sequence Analysis Suite (Biomax Informatics AG, Martinsried, Germany).Gene models constructed by the software were used as a starting pointfor detecting GH61 homologues in the genome. More precise gene modelswere constructed manually using multiple known GH61 protein sequences asa guide.

To generate genomic DNA for PCR amplification, Thielavia terrestris NRRL8126 was grown in 50 ml of NNCYP medium supplemented with 1% glucose ina baffled shake flask at 42° C. and 200 rpm for 24 hours. Mycelia wereharvested by filtration, washed twice in TE (10 mM Tris-1 mM EDTA), andfrozen under liquid nitrogen. A pea-size piece of frozen mycelia wassuspended in 0.7 ml of 1% lithium dodecyl sulfate in TE and disrupted byagitation with an equal volume of 0.1 mm zirconia/silica beads (BiospecProducts, Inc., Bartlesville, Okla., USA) for 45 seconds in a FastPrepFP120 (ThermoSavant, Holbrook, N.Y., USA). Debris was removed bycentrifugation at 13,000×g for 10 minutes and the cleared supernatantwas brought to 2.5 M ammonium acetate and incubated on ice for 20minutes. After the incubation period, the nucleic acids wereprecipitated by addition of 2 volumes of ethanol. After centrifugationfor 15 minutes in a microfuge at 4° C., the pellet was washed in 70%ethanol and air dried. The DNA was resuspended in 120 μl of 0.1×TE andincubated with 1 μl of DNase-free RNase A at 37° C. for 20 minutes.Ammonium acetate was added to 2.5 M and the DNA was precipitated with 2volumes of ethanol. The pellet was washed in 70% ethanol, air dried, andresuspended in TE buffer.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH61N gene from the genomic DNA.An IN-FUSION™ Cloning Kit was used to clone the fragment directly intothe expression vector, pAILo2 (WO 2005/074647), without the need forrestriction digests and ligation.

Forward primer: (SEQ ID NO: 127) 5′-ACTGGATTTACCATGCCTTCTTTCGCCTCCAA-3′Reverse primer: (SEQ ID NO: 128)5′-TCACCTCTAGTTAATTAATCAGTTTGCCTCCTCAGCCC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 1 μg of Thielavia terrestris genomic DNA, 1× ADVANTAGE®GC-Melt LA Buffer (BD Biosciences, Palo Alto, Calif., USA), 1 μl of 10mM blend of dATP, dTTP, dGTP, and dCTP, 1.25 units of ADVANTAGE® GCGenomic LA Polymerase Mix (BD Biosciences, Palo Alto, Calif., USA), in afinal volume of 25 μl. The amplification conditions were one cycle at94° C. for 1 minute; and 30 cycles each at 94° C. for 30 seconds, 60.5°C. for 30 seconds, and 72° C. for 1 minute. The heat block was then heldat 72° C. for 5 minutes followed by a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 1.1 kb product band was excisedfrom the gel and purified using a MINELUTE® Gel Extraction Kit (QIAGENInc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The fragment was then cloned into pAILo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by 1.0% agarose gel electrophoresis using TAE buffer, excisedfrom the gel, and purified using a QIAQUICK® Gel Extraction Kit (QIAGENInc., Valencia, Calif., USA). The gene fragment and the digested vectorwere combined together in a reaction resulting in the expression plasmidpAG66, in which transcription of the Family GH61N gene was under thecontrol of the NA2-tpi promoter (a modified promoter from the geneencoding neutral alpha-amylase in Aspergillus niger in which theuntranslated leader has been replaced by an untranslated leader from thegene encoding triose phosphate isomerase in Aspergillus nidulans). Therecombination reaction (20 μl) was composed of 1× IN-FUSION™ Buffer (BDBiosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto,Calif., USA), 1 μl of IN-FUSION™ enzyme (diluted 1:10) (BD Biosciences,Palo Alto, Calif., USA), 186 ng of pAILo2 digested with Nco I and Pac I,and 96.6 ng of the Thielavia terrestris GH61N purified PCR product. Thereaction was incubated at 37° C. for 15 minutes followed by 15 minutesat 50° C. The reaction was diluted with 40 μl of TE buffer and 2.5 μl ofthe diluted reaction was used to transform E. coli TOP10 Competentcells. An E. coli transformant containing pAG66 (GH61N gene) wasidentified by restriction enzyme digestion and plasmid DNA was preparedusing a BIOROBOT® 9600.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof pAG43 was used to transform Aspergillus oryzae JaL355. Threetransformants were isolated to individual PDA plates.

Plugs were taken from the original transformation plate of each of thethree transformants and added separately to 1 ml of M410 medium in 24well plates, which were incubated at 34° C. Five days after incubation,7.5 μl of supernatant from each culture was analyzed using CRITERION®stain-free, 8-16% gradient SDS-PAGE, (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) according to the manufacturer's instructions.SDS-PAGE profiles of the cultures showed that several transformants hadnew major bands of approximately 70 kDa and 35 kDa.

Confluent PDA plates of two of the transformants were washed with 5 mlof 0.01% TWEEN® 20 and inoculated into five 500 ml Erlenmeyer flaskcontaining 100 ml of M410 medium and incubated to generate broth forcharacterization of the enzyme. The flasks were harvested on days 3 and5 and filtered using a 0.22 μm stericup suction filter (Millipore,Bedford, Mass.).

Example 23: Preparation of Aspergillus aculeatus CBS 101.43 GH10Xylanase 11

Aspergillus aculeatus CBS 101.43 GH10 xylanase II (SEQ ID NO: 45 [DNAsequence] and SEQ ID NO: 46 [deduced amino acid sequence]) was purifiedfrom SHEARZYME® 2X-CDN01013. The sample was desalted andbuffer-exchanged in 20 mM Bis-Tris pH 6.0 using a HIPREP® 26/10desalting column according to the manufacturer's instructions. Thebuffer exchanged sample was applied to a Q SEPHAROSE® Big Beads column(64 ml) equilibrated with 20 mM Bis-Tris pH 6.0, and the bound proteinwas eluted with a gradient from 0 to 500 mM sodium chloride over 10column volumes. Fractions were pooled and concentrated into 200 mMsodium chloride-20 mM Bis-Tris pH 6.0. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit with bovine serumalbumin as a protein standard.

Example 24: Preparation of Aspergillus fumigatus NN055679 GH10 Xylanase

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (SEQ ID NO: 47 [DNAsequence] and SEQ ID NO: 48 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2(WO 2000/39322) as a host.

The filtered broth was desalted and buffer-exchanged into 20 mM Tris−150 mM NaCl pH 8.5 using a HIPREP® 26/10 Desalting Column according tothe manufacturer's instructions. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit with bovine serum albumin as aprotein standard.

Example 25: Preparation of Trichophaea saccata CBS 804.70 GH10 Xylanase

Trichophaea saccata CBS 804.70 was inoculated onto a PDA plate andincubated for 7 days at 28° C. Several mycelia-PDA agar plugs wereinoculated into 750 ml shake flasks containing 100 ml of MEX-1 medium.The flasks were agitated at 150 rpm for 9 days at 37° C. The fungalmycelia were harvested by filtration through MIRACLOTH® (Calbiochem, SanDiego, Calif., USA) before being frozen in liquid nitrogen. The myceliawere then pulverized into a powder by milling the frozen myceliatogether with an equal volume of dry ice in a coffee grinder precooledwith liquid nitrogen. The powder was transferred into a liquid nitrogenprechilled mortor and pestle and ground to a fine powder with a smallamount of baked quartz sand. The powdered mycelial material was kept at−80° C. until use.

Total RNA was prepared from the frozen, powdered mycelium of Trichophaeasaccata CBS 804.70 by extraction with guanidium thiocyanate followed byultracentrifugation through a 5.7 M CsCl cushion according to Chirgwinet al., 1979, Biochemistry 18: 5294-5299. The polyA enriched RNA wasisolated by oligo (dT)-cellulose affinity chromatography according toAviv et al., 1972, Proc. Natl. Acad. Sci. USA 69: 1408-1412.

Double stranded cDNA was synthesized according to the general methods ofGubler and Hoffman, 1983, Gene 25: 263-269; Sambrook, J., Fritsch, E.F., and Maniantis, T. Molecular cloning: A Laboratory Manual, 2nd ed.,1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Kofodet al., 1994, J. Biol. Chem. 269: 29182-29189, using a polyA-Not Iprimer (Promega Corp., Madison, Wis., USA). After synthesis, the cDNAwas treated with mung bean nuclease, blunt ended with T4 DNA polymerase,and ligated to a 50-fold molar excess of Eco RI adaptors (InvitrogenCorp., Carlsbad, Calif., USA). The cDNA was cleaved with Not I and thecDNA was size fractionated by 0.8% agarose gel electrophoresis using TBEbuffer. The fraction of cDNA of 700 bp and larger was excised from thegel and purified using a GFX® PCR DNA and Gel Band Purification Kitaccording to the manufacturer's instructions.

The directional, size-fractioned cDNA was ligated into Eco RI-Not Icleaved pYES 2.0 (Invitrogen, Carlsbad, Calif., USA). The ligationreactions were performed by incubation at 16° C. for 12 hours, thenheating at 70° C. for 20 minutes, and finally addition of 10 μl of waterto each tube. One μl of each ligation mixture was electroporated into 40μl of electrocompetent E. coli DH10B cells (Invitrogen Corp., Carlsbad,Calif., USA) as described by Sambrook et al., 1989, supra.

The Trichophaea saccata CBS 804.70 library was established in E. coliconsisting of pools. Each pool was made by spreading transformed E. colion LB ampicillin plates, yielding 15,000-30,000 colonies/plate afterincubation at 37° C. for 24 hours. Twenty ml of LB medium was added tothe plate and the cells were suspended therein. The cell suspension wasshaken in a 50 ml tube for 1 hour at 37° C.

Plasmid DNA from several of the library pools of T. saccata CBS 804.70was isolated using a Midi Plasmid Kit (QIAGEN Inc., Valencia, Calif.,USA), according to the manufacturer's instructions, and stored at −20°C.

One μl aliquots of purified plasmid DNA from several of the librarypools were transformed into S. cerevisiae W3124 by electroporation(Becker and Guarante, 1991, Methods Enzymol. 194: 182-187) and thetransformants were plated onto SC-agar plates containing 2% glucose andincubated at 30° C. In total, 50-100 plates containing 250-400 yeastcolonies were obtained from each pool. After 3-5 days of incubation, theSC agar plates were replica plated onto a set of 0.1% AZCL xylan(oat)SC-URA agar plates with galactose. The plates were incubated for2-4 days at 30° C. and xylanase positive colonies were identified ascolonies surrounded by a blue halo. The positive clones werestreak-purified and obtai strain CBS 521.95 ned as single colonies.

Xylanase-expressing yeast colonies were inoculated into 5 ml of YPDmedium in 25 ml tubes. The tubes were shaken overnight at 30° C. One mlof the culture was centrifugated to pellet the yeast cells.

DNA was isolated according to WO 94/14953 and dissolved in 50 μl ofwater. The DNA was transformed into E. coli DH10B using standardprocedures (Sambrook et al., 1989, supra).

Plasmid DNA was isolated from the E. coli transformants using standardprocedures (Sambrook et al., 1989, supra). Plasmids were sequenced usingboth pYES primers as sequencing primers. One specific plasmid clone of1283 bp designated TF12Xy1170 was found to encode a Family 10 glycosidehydrolase protein and was further characterized. More reliable sequencewas obtained by further sequencing of the fragment using the specificprimers shown below designed based on the initial sequence:

TF12Xyl170F1: (SEQ ID NO: 129) 5′-TGAAATGGGATGCTACTGA-3′ TF12Xyl170F2:(SEQ ID NO: 130) 5′-CAACGACTACAACATCGAGG-3′ TF12Xyl170R1:(SEQ ID NO: 131) 5′-ATTTGCTGTCCACCAGTGAA-3′

One plasmid matching the original cDNA sequence was designatedpTF12Xy1170 and the E. coli strain containing this clone was designatedE. coli pTF12Xy1170 and deposited on Jul. 28, 2009, with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, Peoria, Ill., USA, and given the accessionnumber NRRL B-50309.

The nucleotide sequence and deduced amino acid sequence of theTrichophaea saccata gh10a gene are shown in SEQ ID NO: 49 and SEQ ID NO:50, respectively. The coding sequence is 1197 bp including the stopcodon. The encoded predicted protein contains 398 amino acids. The % G+Cof the coding region of the gene is 53.6% and the mature polypeptidecoding region is also 53.6%. Using the SignalP program, version 3(Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal peptide of19 residues was predicted. The predicted mature protein contains 379amino acids with a molecular mass of 40.4 kDa.

Analysis of the deduced amino acid sequence of the gh10a gene with theInterproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17:847-848) showed that the GH10A protein contained the core sequencetypical of a Family 10 glycoside hydrolase, extending from approximatelyamino acid residue 65 to residue 377 of the predicted maturepolypeptide. The GH10A protein also contained the sequence signature ofa type 1 fungal cellulose binding domain (CBMI). This sequence signatureknown as Prosite Entry PS00562 (Sigrist et al., 2002, Brief Bioinform.3: 265-274) was present from amino acid residue 8 to residue 35 of thepredicted mature polypeptide.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Trichophaea saccata gene encoding the GH10A maturepolypeptide shared 62.6% and 62.0% identity (excluding gaps) to thededuced amino acid sequences of Family 10 glycoside hydrolase proteinsfrom Phanerochaete chrysosporium and Meripilus giganteus, respectively(accession numbers UNIPROT:B7SIW2 and GENESEQP:AAW23327, respectively).

The Trichophaea saccata CBS 804.70 gh10a gene was excised from thepTF12xyl170 using Bam HI and Xho 1, and ligated into the Aspergillusexpression vector pDAu109 (WO 2005/042735), also digested with Bam HIand Xho 1, using standard methods (Sambrook et al., 1989, supra). Theligation reaction was transformed into E. coli TOP10 chemicallycompetent cells according to the manufacturer's instructions. Eightcolonies were grown overnight in LB ampicillin medium and plasmid DNAwas isolated using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA) according to the manufacturer's directions. Plasmidscontaining the correct size inserts were sequenced to determineintegrity and orientation of the insert. Plasmid pDAu81 #5 was found tobe error free and was therefore chosen for scale-up.

Protoplasts of Aspergillus oryzae BECH2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. A. oryzae BECh2 was constructed as describedin WO 00139322. One hundred microliters of protoplast suspension weremixed with 5-25 μg of the Aspergillus expression vector pDAu81 #5 in 10μl of STC composed of 1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mMCaCl₂ (Christensen et al., 1988, Bio/Technology 6: 1419-1422). Themixture was left at room temperature for 25 minutes. Two hundredmicroliters of 60% PEG 4000 (BDH, Poole, England) (polyethylene glycol,molecular weight 4,000), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 wereadded and gently mixed and finally 0.85 ml of the same solution wasadded and gently mixed. The mixture was left at room temperature for 25minutes, centrifuged at 2,500×g for 15 minutes, and the pellet wasresuspended in 2 ml of 1.2 M sorbitol. This sedimentation process wasrepeated, and the protoplasts were spread on COVE plates. Afterincubation for 4-7 days at 37° C. spores were picked and spread on COVEplates containing 0.01% TRITON® X-100 in order to isolate singlecolonies. The spreading was repeated twice more on COVE sucrose medium(Cove, 1996, Biochim. Biophys. Acta 133: 51-56) containing 1 M sucroseand 10 mM sodium nitrate.

Ten of the transformants were inoculated in 10 ml of YPG medium. After3-4 days of incubation at 30° C., 200 rpm, the supernatant was removedand analyzed by SDS-PAGE 10% Bis-Tris gels (Invitrogen, Carlsbad,Calif., USA) as recommended by the manufacturer. Gels were stained withCoomassie blue and all isolates displayed a diffuse band between 35 and45 kDa. These transformants were analyzed further for xylanase activityat pH 6.0 using a modified AZCL-arabinoxylan as substrate isolated fromwheat (Megazyme, Wicklow, Ireland) in 0.2 M sodium phosphate pH 6.0buffer containing 0.01% TRITON® X-100 according to the manufacturer'sinstructions. The transformant producing the highest level of activitywas chosen for production of the xylanase.

The transformant producing the highest level of activity was grown usingstandard methods. The broth was filtered using Whatmann glass filtersGF/D, GF/A, GF/C, GF/F (2.7 μm, 1.6 μm, 1.2 μm and 0.7 μm, respectively)(Whatman, Piscataway, N.J., USA) followed by filtration using a NALGENE®bottle top 0.45 μm filter (Thermo Fisher Scientific, Rochester, N.Y.,USA).

Ammonia sulfate was added to the filtered broth to a final concentrationof 3 M and the precipitate was collected after centrifugation at10,000×g for 30 minutes. The precipitate was dissolved in 10 mM Tris/HClpH 8.0 and dialyzed against 10 mM Tris/HCl pH 8.0 overnight. Thedialyzed preparation was applied to a 150 ml Q SEPHAROSE® Fast Flowcolumn equilibrated with 10 mM Tris/HCl pH 8.0 and the enzyme was elutedwith a 1050 ml (7 column volumes) linear salt gradient from 0 to 1 MNaCl in 10 mM Tris/HCl pH 8.0. Elution was followed at 280 nm andfractions were collected and assayed for xylanase activity using 0.2%AZCL-Arabinoxylan from wheat in 0.2 M sodium phosphate buffer pH 6.0containing 0.01% TRITON® X-100. Fractions containing xylanase activitywere pooled and stored at −20° C.

Example 26: Preparation of Penicillium pinophilum GH10 Xylanase

Penicillium pinophilum GH10 xylanase (SEQ ID NO: 51 [DNA sequence] andSEQ ID NO: 52 [deduced amino acid sequence]) was prepared according tothe following procedure. Compost samples were collected from Yunnan,China on Dec. 12, 2000.

Penicillium pinophilum NN046877 was isolated using single sporeisolation techniques on PDA plates at 45° C. Penicillium pinophilumstrain NN046877 was inoculated onto a PDA plate and incubated for 4 daysat 37° C. in the darkness. Several mycelia-PDA plugs were inoculatedinto 500 ml shake flasks containing 100 ml of NNCYP-PCS medium. Theflasks were incubated for 5 days at 37° C. with shaking at 160 rpm. Themycelia were collected at day 4 and day 5. The mycelia from each daywere frozen in liquid nitrogen and stored in a −80° C. freezer untiluse.

The frozen mycelia were transferred into a liquid nitrogen prechilledmortar and pestle and ground to a fine powder. Total RNA was preparedfrom the powdered mycelia of each day by extraction with TRIZOL™reagent. The polyA enriched RNA was isolated using a mTRAP™ Total Kit.

Double stranded cDNA from each day was synthesized with a SMART™ cDNAlibrary Construction Kit (Clontech Laboratories, Inc., Mountain View,Calif., USA). The cDNA was cleaved with Sfi I and the cDNA was sizefractionated by 0.8% agarose gel electrophoresis using TBE buffer. Thefraction of cDNA of 500 bp and larger was excised from the gel andpurified using a GFX® PCR DNA and Gel Band Purification Kit according tothe manufacturer's instructions. Then equal amounts of cDNA from day 4and day 5 were pooled for library construction.

The pooled cDNA was then directionally cloned by ligation into Sfi Icleaved pMHas7 (WO 2009/037253) using T4 ligase (New England Biolabs,Inc., Beverly, Mass., USA) according to the manufacturer's instructions.The ligation mixture was electroporated into E. coli ELECTROMAX™ DH10B™cells (Invitrogen Corp., Carlsbad, Calif., USA) using a GENE PULSER® andPulse Controller (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) at25 uF, 25 mAmp, 1.8 kV with a 1 mm gap cuvette according to themanufacturer's procedure.

The electroporated cells were plated onto LB plates supplemented with 50mg of kanamycin per liter. A cDNA plasmid pool was prepared from 60,000total transformants of the original pMHas7 vector ligation. Plasmid DNAwas prepared directly from the pool of colonies using a QIAGEN® PlasmidKit (QIAGEN Inc., Valencia, Calif., USA).

A transposon containing plasmid designated pSigA4 was constructed fromthe pSigA2 transposon containing plasmid described WO 2001/77315 inorder to create an improved version of the signal trapping transposon ofpSigA2 with decreased selection background. The pSigA2 transposoncontains a signal less beta-lactamase construct encoded on thetransposon itself. PCR was used to create a deletion of the intact betalactamase gene found on the plasmid backbone using a proofreading PfuTurbo polymerase PROOFSTART™ (QIAGEN GmbH Corporation, Hilden, Germany)and the following 5′ phosphorylated primers (TAG Copenhagen, Denmark):

SigA2NotU-P: (SEQ ID NO: 132) 5′-TCGCGATCCGTTTTCGCATTTATCGTGAAACGCT-3′SigA2NotD-P: (SEQ ID NO: 133) 5′-CCGCAAACGCTGGTGAAAGTAAAAGATGCTGAA-3′

The amplification reaction was composed of 1 μl of pSigA2 (10 ng/μl), 5μl of 10× PROOFSTART™ Buffer (QIAGEN GmbH Corporation, Hilden, Germany),2.5 μl of dNTP mix (20 mM), 0.5 μl of SigA2NotU-P (10 mM), 0.5 μl ofSigA2NotD-P (10 mM), 10 μl of Q solution (QIAGEN GmbH Corporation,Hilden, Germany), and 31.25 μl of deionized water. A DNA ENGINE™ ThermalCycler (MJ Research Inc., Waltham, Mass., USA) was used foramplification programmed for one cycle at 95° C. for 5 minutes; and 20cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for 4 minutes.

A 3.9 kb PCR reaction product was isolated by 0.8% agarose gelelectrophoresis using TAE buffer and 0.1 μg of ethidium bromide per ml.The DNA band was visualized with the aid of an EAGLE EYE® Imaging System(Stratagene, La Jolla, Calif., USA) at 360 nm. The 3.9 kb DNA band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 3.9 kb fragment was self-ligated at 16° C. overnight with 10 unitsof T4 DNA ligase (New England Biolabs, Inc., Beverly, Mass., USA), 9 μlof the 3.9 kb PCR fragment, and 1 μl of 10× ligation buffer (New EnglandBiolabs, Inc., Beverly, Mass., USA). The ligation was heat inactivatedfor 10 minutes at 65° C. and then digested with Dpn I at 37° C. for 2hours. After incubation, the digestion was purified using a GFX® PCR DNAand Gel Band Purification Kit.

The purified material was then transformed into E. coli TOP10 competentcells according to the manufacturer's instructions. The transformationmixture was plated onto LB plates supplemented with 25 μg ofchloramphenicol per ml. Plasmid minipreps were prepared from severaltransformants and digested with Bgl II. One plasmid with the correctconstruction was chosen. The plasmid was designated pSigA4. PlasmidpSigA4 contains the Bgl II flanked transposon SigA2 identical to thatdisclosed in WO 2001/77315.

A 60 μl sample of plasmid pSigA4 DNA (0.3 μg/μl) was digested with BglII and separated by 0.8% agarose gel electrophoresis using TBE buffer. ASigA2 transposon DNA band of 2 kb was eluted with 200 μl of EB buffer(QIAGEN GmbH Corporation, Hilden, Germany) and purified using a GFX® PCRDNA and Gel Band Purification Kit according to the manufacturer'sinstructions and eluted in 200 μl of EB buffer. SigA2 was used fortransposon assisted signal trapping.

A complete description of transposon assisted signal trapping isdescribed in WO 2001/77315. The plasmid pool was treated with transposonSigA2 and HYPERMU™ transposase (EPICENTRE Biotechnologies, Madison,Wis., USA) according to the manufacturer's instructions.

For in vitro transposon tagging of the Penicillium pinophilum cDNAlibrary, 2 μl of SigA2 transposon containing approximately 100 ng of DNAwere mixed with 1 μl of the plasmid DNA pool of the Penicilliumpinophilum cDNA library containing 1 μg of DNA, 1 μl of HYPERMU™transposase, and 2 μl of 10× buffer (EPICENTRE Biotechnologies, Madison,Wis., USA) in a total volume of 20 μl and incubated at 30° C. for 3hours followed by adding 2 μl of stop buffer (EPICENTRE Biotechnologies,Madison, Wis., USA) and heat inactivation at 75° C. for 10 minutes. TheDNA was precipitated by addition of 2 μl of 3 M sodium acetate pH 5 and55 μl of 96% ethanol and centrifuged for 30 minutes at 10,000×g, 4° C.The pellet was washed in 70% ethanol, air dried at room temperature, andresuspended in 10 μl of deionized water.

A 2 μl volume of the transposon tagged plasmid pool was electroporatedinto 50 μl of E. coli ELECTROMAX™ DH10B™ cells (Invitrogen Corp.,Carlsbad, Calif., USA) according to the manufacturer's instructionsusing a GENE PULSER® and Pulse Controller at 25 uF, 25 mAmp, 1.8 kV witha 1 mm gap cuvette according to the manufacturer's procedure.

The electroporated cells were incubated in SOC medium with shaking at225 rpm for 1 hour at 37° C. before being plated onto the followingselective media: LB medium supplemented with 50 μg of kanamycin per ml;LB medium supplemented with 50 μg of kanamycin per ml and 15 μg ofchloramphencol per ml; and LB medium supplemented with 50 μg ofkanamycin per ml, 15 μg of chloramphencol per ml, and 30 μg ofampicillin per ml.

From plating of the electroporation onto LB medium supplemented withkanamycin, chloramphencol and ampicillin, approximately 200 colonies per50 μl were observed after 3 days at 30° C. All colonies were replicaplated onto LB kanamycin, chloramphenicol, and ampicillin mediumdescribed above. Five hundred colonies were recovered under thisselection condition. The DNA from each colony was sequenced with thetransposon forward and reverse primers (primers A and B), shown below,according to the procedure disclosed in WO 2001/77315 (page 28).

Primer A: (SEQ ID NO: 134) 5′-agcgtttgcggccgcgatcc-3′ Primer B:(SEQ ID NO: 135) 5′-ttattcggtcgaaaaggatcc-3′

DNA sequences were obtained from SinoGenoMax Co., Ltd (Beijing, China).Primer A and primer B sequence reads for each plasmid were trimmed toremove vector and transposon sequence. The assembled sequences weregrouped into contigs by using the program PhredPhrap (Ewing et al.,1998, Genome Research 8: 175-185; Ewing and Green, 1998, Genome Research8: 186-194). All contigs were subsequently compared to sequencesavailable in standard public DNA and protein sequences databases(TrEMBL, SWALL, PDB, EnsemblPep, GeneSeqP) using the program BLASTX2.0a19MP-WashU [14 Jul. 1998] [Build linux-x86 18:51:44 30 Jul. 1998](Gish et al., 1993, Nat. Genet. 3: 266-72). The family GH10 xylanasecandidate was identified directly by analysis of the BlastX results.

Penicillium pinophilum NN046877 was grown on a PDA agar plate at 37° C.for 4-5 days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Based on the Penicillium pinophilum GH10 xylanase gene informationobtained as described above, oligonucleotide primers, shown below, weredesigned to amplify the GH61 gene from genomic DNA of Penicilliumpinophilum GH10 NN046877. An IN-FUSION® CF Dry-down Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) was used toclone the fragment directly into the expression vector pPFJO355, withoutthe need for restriction digestion and ligation.

Sense primer: (SEQ ID NO: 136) 5′-ACACAACTGGGGATCCACCATGACTCTAGTAAAGGCTATTCTTTTAGC- 3′ Antisense primer:(SEQ ID NO: 137) 5′-GTCACCCTCTAGATCTTCACAAACATTGGGAGTAGTATGG-3′Bold letters represented the coding sequence and the remaining sequencewas homologous to insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium sp. NN051602 genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and0.6 unit of PHUSION™ High-Fidelity DNA Polymerase, in a final volume of50 μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minutes; 5 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 56° C. for 30 seconds,with a 1° C. increase per cycle and elongation at 72° C. for 75 seconds;and 25 cycles each at 98° C. for 15 seconds, 65C for 30 seconds and 72°C. for 75 seconds; and a final extension at 72° C. for 10 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.4 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION® CF Dry-down PCR Cloning Kit resulting in pPpin3 in whichtranscription of the Penicillium pinophilum GH10 xylanase gene was underthe control of a promoter from the gene for Aspergillus oryzaealpha-amylase. In brief, 30 ng of pPFJO355 digested with Barn I and BglII, and 60 ng of the Penicillium pinophilum GH10 xylanase gene purifiedPCR product were added to a reaction vial and resuspended in a finalvolume of 10 μl with addition of deionized water. The reaction wasincubated at 37° C. for 15 minutes and then 50° C. for 15 minutes. Threeμl of the reaction were used to transform E. coli TOP10 competent cells.An E. coli transformant containing pPpin3 was detected by colony PCR andplasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc.,Valencia, Calif., USA). The Penicillium pinophilum GH10 xylanase geneinsert in pPpin3 was confirmed by DNA sequencing using a 3730XL DNAAnalyzer.

The same PCR fragment was cloned into vector pGEM-T using a pGEM-TVector System to generate pGEM-T-Ppin3. The Penicillium pinophilum GH10xylanase gene contained in pGEM-T-Ppin3 was confirmed by DNA sequencingusing a 3730XL DNA Analyzer. E. coli strain T-Ppin3, containingpGEM-T-Ppin3, was deposited with the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSM), D-38124 Braunschweig,Germany on Sep. 7, 2009, and assigned accession number DSM 22922.

DNA sequencing of the Penicillium pinophilum genomic clone encoding aGH10 polypeptide having xylanase activity was performed with an AppliedBiosystems Model 3700 Automated DNA Sequencer using version 3.1 BIG-DYE™terminator chemistry (Applied Biosystems, Inc., Foster City, Calif.,USA) and dGTP chemistry (Applied Biosystems, Inc., Foster City, Calif.,USA) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were compared to each otherwith assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of thePenicillium pinophilum gh10 gene are shown in SEQ ID NO: 51 and SEQ IDNO: 52, respectively. The coding sequence is 1442 bp including the stopcodon and is interrupted by three introns of 51 bp (199-249), 73 bp(383-455), and 94 bp (570-663). The encoded predicted protein is 407amino acids. The % G+C of the coding sequence of the gene (includingintrons) is 47.99% G+C and the mature polypeptide coding sequence is49.22%. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 388 amino acids with a predictedmolecular mass of 41.5 kDa and an isoelectric pH of 5.03.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Penicillium pinophilum gene encoding the GH10polypeptide having xylanase activity shares 76% and 87% identity(excluding gaps) to the deduced amino acid sequence of a predicted GH10family protein from Talaromyces emersonii (AAU99346) and Penicilliummarneffei (B6QN64), respectively.

Aspergillus oryzae HowB101 (WO 95/35385 Example 1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Three μg of pPpin3 were transformed intoAspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pPpin3 yieldedabout 50 transformants. Twelve transformants were isolated to individualMinimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C. with shaking at 150 rpm. After 3days incubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT® Blue (Expedeon Ltd., Babraham Cambridge, UK).SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 55 kDa. The expressionstrain was designated Aspergillus oryzae EXP02765.

A slant of one transformant, designated transformant 2, was washed with10 ml of YPM and inoculated into a 2 liter flask containing 400 ml ofYPM medium to generate broth for characterization of the enzyme. Theculture was harvested on day 3 and filtered using a 0.45 μm DURAPORE®Membrane (Millipore, Bedford, Mass., USA).

A 1 liter volume of supernatant of the recombinant Aspergillus oryzaestrain EXP02765 was precipitated with ammonium sulfate (80% saturation)and redissolved in 50 ml of 25 mM sodium acetate pH 4.3, and thendialyzed against the same buffer and filtered through a 0.45 μm filter.The solution was applied to a 40 ml Q SEPHAROSE™ Fast Flow column columnequilibrated in 25 mM sodium acetate pH 4.3. The recombinant GH10protein did not bind to the column. The fractions with xylanase activitywere collected and evaluated by SDS-PAGE as described above. Fractionscontaining a band of approximately 55 kDa were pooled. The pooledsolution was concentrated by ultrafiltration.

Example 27: Preparation of Thielavia terrestris GH10E Xylanase

Thielavia terrestris NRRL 8126 GH10E xylanase (SEQ ID NO: 53 [DNAsequence] and SEQ ID NO: 54 [deduced amino acid sequence]) was preparedaccording to the following procedure.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris gh10e gene from genomic DNA. AnInFusion Cloning Kit (Clontech, Mountain View, Calif.) was used to clonethe fragment directly into the expression vector, pAILo2 (WO2005/074647).

Forward primer: (SEQ ID NO: 138)5′-ACTGGATTTACCATGGCCCTCAAATCGCTCCTGTTG-3′ Reverse primer:(SEQ ID NO: 139) 5′-TCACCTCTAGTTAATTAATTACAAGCACTGAGAGTA-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 2 μg of Thielavia terrestris genomic DNA, 1×Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 2×PCR, Enhancer solution(Invitrogen, Carlsbad, Calif., USA), 1.5 μl of 10 mM blend of dATP,dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA Polymerase, and 1μl of 50 mM MgSO₄ in a final volume of 50 μl. The amplificationconditions were one cycle at 94° C. for 2 minutes; and 30 cycles each at94° C. for 15 seconds, 59.5° C. for 30 seconds, and 68° C. for 150seconds. The heat block was then held at 68° C. for 7 minutes followedby a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer and an approximately 1.2 kb product band was excised from the geland purified using a MINELUTE® Gel Extraction Kit (QIAGEN Inc.,Valencia, Calif., USA) according to the manufacturer's instructions.

The fragment was then cloned into pAILo2 using an IN-FUSION™ CloningKit. The vector was digested with Nco I and Pac I (using conditionsspecified by the manufacturer). The fragment was purified by gelelectrophoresis as above and a QIAQUICK® Gel Extraction Kit. The genefragment and the digested vector were combined together in a reactionresulting in the expression plasmid pAG29, in which transcription of thegh10e gene was under the control of the NA2-tpi promoter (a hybrid ofthe promoters from the genes for Aspergillus niger neutral alpha-amylaseand Aspergillus nidulans triose phosphate isomerase). The recombinationreaction (10 μl) was composed of 1× InFusion Buffer (Clontech, MountainView, Calif., USA), 1×BSA (Clontech, Mountain View, Calif., USA), 0.5 μlof InFusion enzyme (diluted 1:10) (Clontech, Mountain View, Calif.,USA), 93 ng of pAILo2 digested with Nco I and Pac I, and 1 μl of theThielavia terrestris gh10e purified PCR product. The reaction wasincubated at 37° C. for 15 minutes followed by 15 minutes at 50° C. Thereaction was diluted with 40 μl of TE buffer and 2.5 μl of the dilutedreaction was used to transform E. coli SOLOPACK® Gold cells.

Plasmid DNA was prepared using a BIOROBOT® 9600 and a restriction enzymedigest performed. Putative pAG29 clones were digested with Pst I. Theplasmid DNA from these clones was then sequenced to identify cloneswithout PCR induced errors. Sequencing reactions contained 1.5 μl ofplasmid DNA, 4.5 μl of water, and 4 μl of sequencing master-mixcontaining 1 μl of 5× sequencing buffer (Millipore, Billerica, Mass.,USA), 1 μl of BIGDYE™ terminator (Applied Biosystems, Inc., Foster City,Calif., USA), 1 μl of water and one of the following primers at 3.2μmoles per reaction.

pAlLo2 5′ (SEQ ID NO: 140) 5′-TGTCCCTTGTCGATGCG-3′ pAlLo2 3′(SEQ ID NO: 141) 5′-CACATGACTTGGCTTCC-3′

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof pAG29 was used to transform Aspergillus oryzae JaL355. Twenty-fourtransformants were isolated to individual PDA plates.

Confluent PDA plates of 24 transformants were washed with 5 ml of 0.01%TWEEN® 20 and spores collected. Eight μl of each spore stock was addedto 1 ml of YPG, YPM, and M410 separately in 24 well plates and incubatedat 34° C. Three days after incubation, 7.5 μl of supernatant fromselected culture was analyzed using Criterion stain-free, 8-16% gradientSDS-PAGE, (BioRad, Hercules, Calif.) according to the manufacturer'sinstructions. SDS-PAGE profiles of the cultures showed that severaltransformants had a new major band of approximately 50 kDa and the bestexpression in M410. After a total of six days of incubation, all M410cultures were sampled as described above and analyzed using Criterionstain-free, 8-16% gradient SDS-PAGE gel, (BioRad, Hercules, Calif.) atwhich point the transformant exhibiting the best expression wasselected.

A confluent PDA plate of the top transformant was washed with 5 ml of0.01% TWEEN® 20 and inoculated into five 500 ml Erlenmeyer flaskcontaining 100 ml of M410 medium to generate broth for characterizationof the enzyme. The flasks were harvested on day 5. Broths were filteredusing a 0.22 μm stericup suction filter (Millipore, Bedford, Mass.).

Example 28: Preparation of Thermobifida fusca GH11 Xylanase

A linear integration vector-system was used for the expression cloningof a Thermobifida fusca DSM 22883 GH11 xylanase gene (SEQ ID NO: 55 [DNAsequence] and SEQ ID NO: 56 [deduced amino acid sequence]). The linearintegration construct was a PCR fusion product made by fusion of eachgene between two Bacillus subtils homologous chromosomal regions alongwith a strong promoter and a chloramphenicol resistance marker. Thefusion was made by SOE PCR (Horton et al., 1989, Gene 77: 61-68). TheSOE PCR method is also described in WO 2003/095658. Each gene wasexpressed under the control of a triple promoter system (as described inWO 99/43835), consisting of the promoters from Bacillus licheniformisalpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), and the Bacillus thuringiensis cryIIIA promoter includingstabilizing sequence. The gene coding for chloramphenicolacetyl-transferase was used as marker (Diderichsen et al., 1993, Plasmid30: 312). The final gene construct was integrated into the Bacilluschromosome by homologous recombination into the pectate lyase locus.

The GH11 xylanase gene (SEQ ID NO: 55 [DNA sequence] and SEQ ID NO: 56[deduced amino acid sequence]) was isolated from Thermobifida fusca DSM22883 (NN018438) by a polymerase chain reaction (PCR1) using the primersshown in the table below. The primers are based on the protein sequenceUNIPROT:Q5RZ98. Thermobifida fusca DSM 22883 was isolated from a soilsample obtained from Oahu, Hi. in 2001. The xylanase gene was cloned asa full-length gene and as a truncated gene. The genes were designed tocontain a C-terminal HQHQHQH tag to ease purification but the His-tagwas not used for the purification. The forward primer Ocs3 was designedso the gene was amplified from the start codon (ATG) and has 24 basesoverhang (shown in italic in the table below). This overhang iscomplementary to part of one of the two linear vector fragments and isused when the PCR fragment and the vector fragments are assembled(described below). The reverse primer Ocs1 was designed to amplify thetruncated version of the gene while the reverse primer Ocs2 was designedto amplify the full-length gene. Both Ocs1 and Ocs2 carry an overhangconsisting of 24 bp encoding a HQHQHQH-tag and a stop codon (theoverhang is shown in italic in the table below). This overhang iscomplementary to part of one of the two linear vector fragments and isused when the PCR fragment and the vector fragments are assembled(described below).

A PCR fragment was isolated containing the full-length xylanase gene andthe short 24 bp flanking DNA sequences included in the primers asoverhang. Another PCR fragment was isolated containing the truncatedxylanase gene and the same short 24 bp flanking DNA sequences.

For each gene construct 3 fragments were PCR amplified: the genefragment from genomic DNA from the Thermobifida fusca (NN018438), theupstream flanking fragment was amplified with primers 260558 andiMB1361Uni1, and the downstream flanking fragment was amplified withprimers 260559 and oth432 from genomic DNA of the strain iMB1361(described in patent application WO 2003095658). All primers used arelisted in Table below.

Amplification of SPECIFIC PRIMER FORWARD SPECIFIC PRIMER REVERSEFull-length gene OCS3: OCS2: 5′-

5′-

ATGAACCATGCCCCCG

GTTGGCGCTGCAGGA CCA-3′ (SEQ ID NO: 142) CACCGT-3′ (SEQ ID NO: 143)Truncated gene OCS3: OCS1: 5′-

5′-

ATGAACCATGCCCCCG

GGGGTTGTCACCGCC CCA 3′ (SEQ ID NO: 142) GCT-3′ (SEQ ID NO: 144) Upstream260558: iMB1361Uni1: flanking 5′-GAGTATCGCCAGTAAGGGG5′-TCTTTATCCTCTCCTTTTTTT fragment CG 3′ (SEQ ID NO: 145)CAGAGCTC 3′ (SEQ ID NO: 146) Downstream oth432: 260559: flanking5′-CATCAGCACCAACACCAGCA 5′-GCAGCCCTAAAATCGCATAA fragmentTCCGTAATCGCATGTTCAATCC AGC-3′ (SEQ ID NO: 148) GCTCCATA 3′ (SEQ ID NO:147)

The gene fragment was amplified using a proofreading polymerase PHUSION™DNA Polymerase according to the manufacturer's instructions with theaddition of 2% DMSO. The two flanking DNA fragments were amplified withan EXPAND™ High Fidelity PCR System according to the manufacturer'srecommendations. The PCR conditions were as follows: one cycle at 94° C.for 2 minutes; 10 cycles each at 94° C. for 15 seconds, 50° C. for 45seconds, 68° C. for 4 minutes; 20 cycles each at 94° C. for 15 seconds,50° C. for 45 seconds, and 68° C. for 4 minutes (+20 seconds extensionpr cycle); and one cycle at 68° C. for 10 minutes The 3 PCR fragmentswere subjected to a subsequent splicing by overlap extension (SOE) PCRreaction to assemble the 3 fragments into one linear vector construct.This was performed by mixing the 3 fragments in equal molar ratios and anew PCR reaction was run under the following conditions: one cycle at94° C. for 2 minutes; 10 cycles each at 94° C. for 15 seconds, 50° C.for 45 seconds, and 68° C. for 5 minutes; 10 cycles each at 94° C. for15 seconds, 50° C. for 45 seconds, and 68° C. for 8 minutes; 15 cycleseach at 94° C. for 15 seconds, 50° C. for 45 seconds, and 68° C. for 8minutes (in addition 20 seconds extra per cycle). After the first cyclethe two end primers 260558 and 260559 were added (20 μMol of each). Twoμl of the PCR product was transformed into Bacillus subtilis.Transformants were selected on LB plates supplemented with 6 μg ofchloramphenicol per ml. The full-length xylanase coding sequence wasintegrated by homologous recombination into the genome of the Bacillussubtilis host TH1 (amy-, spo-, apr-, npr-; WO 2005/038024). Thetruncated xylanase coding sequence was integrated by homologousrecombination into the genome of the Bacillus subtilis host PL2317(amy-, spo-, apr-, npr-, xyl-).

Transformants were then screened for their ability to produce largeamounts of active xylanase. The screening was based on intensity of theband corresponding to the heterologous expressed protein by SDS-PAGEanalysis and activity of the enzyme on LB agar plates containingAZCL-xylan (Megazyme International Ireland, Ltd., Wicklow, Ireland).

One transformant, Bacillus subtilis EXP01687, was isolated whichcontained the full-length xylanase gene expressed in the host strainBacillus subtilis TH1. The full-length xylanase gene sequence ofBacillus subtilis EXP01687 was confirmed by Sanger sequencing. Theprotein sequence differs by 2 amino acids from UNIPROT:Q5RZ98.

Another transformant, Bacillus subtilis EXP01672, was isolated whichcontains the truncated xylanase gene expressed in the host strainBacillus subtilis PL2317. The truncated xylanase gene sequence ofBacillus subtilis EXP01672 was confirmed by Sanger sequencing to encodethe secretion signal and mature amino acid sequence to proline atposition 236 of SEQ ID NO: 56. The protein sequence differs by 2 aminoacids from UNIPROT:Q5RZ98.

Bacillus subtilis EXP01687 was grown in 500 ml baffled Erlenmeyer flaskscontaining Cal-18 medium supplemented with 34 mg of chloramphenicol perliter for 2 days at 37° C. with shaking at 200 rpm. The enzyme waspurified from the culture supernatant according to the protocoldescribed below.

In step 1, the whole culture (800 ml) was centrifuged at 17,600×g for 30minutes and then filtered through a SEITZ-EKS filter (Pall SeitzschenkFiltersystems GmbH, Bad Kreuznach, Germany). Sodium chloride was addedto the filtered sample to 50 mM NaCl and the pH was adjusted to pH 7.5.The sample (750 ml) was applied to a 20 ml Ni SEPHAROSE® 6 Fast Flowcolumn (GE Healthcare, Piscataway, N.J., USA) equilibrated with 50 mMNa₂HPO₄, 50 mM NaCl pH 7.5, and the bound protein was eluted with a 5column volume gradient to 100% 50 mM Na₂HPO₄, 500 mM imidazol pH 7.5.The enzyme was found in the effluent by SDS-PAGE analysis revealing aprotein of the correct size in the effluent and nothing of the correctsize in the eluent. This result was confirmed by activity measurementusing AZCL-arabinoxylan (wheat) as substrate as described above.

In step 2, ammonium sulfate was added to the effluent (of step 1) to 1 Mand the pH adjusted to 7.5. The sample (800 ml) was applied to a 60 mlTOYOPEARL® Phenyl-650M column (TOSOH Corporation, Tokyo, Japan)equilibrated with 1 M ammonium sulfate pH 7.5. The bound protein waseluted with Milli-Q® ultrapure water (Millipore, Billerica, Mass., USA).Fractions with A280 were pooled (55 ml). The pooled fractions weredesalted and buffer-exchanged with 25 mM acetic acid pH 4.5 using aHIPREP® 26/10 desalting column according to the manufacturer'sinstructions.

In step 3, the buffer exchanged sample of step 2 (110 ml) was diluted2.5-fold with Milli-Q®ultrapure water and applied to a 10 ml SPSEPHAROSE® Fast Flow column (GE Healthcare, Piscataway, N.J., USA)equilibrated with 25 mM acetic acid pH 4.5. The bound protein was elutedwith a five column volume gradient from 0 to 500 mM sodium chloride in25 mM acetic acid pH 4.5. Based on SDS-PAGE analysis and A280 and A₂₆₀fractions were pooled (10 ml).

In step 4, the pooled fractions of step 3 were diluted 25-fold withMilli-Q® ultrapure water and the pH was adjusted to pH 6.0 and appliedto 10 ml SP SEPHAROSE® Fast Flow column equilibrated with 5 mM succinicacid pH 6.0. The bound protein was eluted with a ten column volumegradient from 0 to 500 mM sodium chloride in 5 mM succinic acid pH 6.0.Based on SDS-PAGE analysis and A₂₈₀ and A₂₆₀ fractions were pooled (12ml). Separate portions of the pooled fractions were then subjected tothree different steps (steps 5(a), 5(b), and 5(c)) described below.

In step 5(a), 6 ml of the pooled fractions of step 4 were diluted to 25ml with Milli-Q® ultrapure water and the pH was adjusted to pH 9.0 andapplied to a 1 ml RESOURCE™ Q column (GE Healthcare, Piscataway, N.J.,USA) equilibrated with 25 mM boric acid pH 9.0. The bound protein waseluted with a ten column volume gradient from 0 to 500 mM sodiumchloride in 25 mM boric acid pH 9.0. Based on A280 and A₂₆₀, the proteinwas in the effluent (35 ml). This sample was concentrated using anAMICON® ultrafiltration cell equipped with a 5 kDa cut-off membrane(Millipore, Billerica, Mass., USA).

In step 5(b), 3 ml of the pool of step 4 were diluted to 30 ml withMilli-Q® ultrapure water and pH was adjusted to pH 9.6 and applied to a1 ml RESOURCE™ Q column equilibrated with 12.5 mM boric acid pH 9.7. Thebound protein was eluted with a ten column volume gradient from 0 to 500mM sodium chloride in 12.5 mM boric acid pH 9.7. Based on A280 and A₂₆₀and SDS-PAGE analysis, the protein was in the effluent (36 ml).

In step 5(c), one quarter of the pool of step 4 was diluted withMilli-Q® ultrapure water and the pH was adjusted to pH 8.0 and appliedto a 1 ml RESOURCE™ Q column equilibrated with 25 mM borate pH 8.0. Thebound protein was eluted with a ten column volume gradient from 0 to 500mM sodium chloride in 25 mM borate pH 8.0. Based on A₂₈₀ and A₂₆₀ andSDS-PAGE analysis, the protein was in the effluent (25 ml).

In step 6, the sample from step 5(a), the effluent from step 5(b), andthe effluent from step 5(c) were pooled and diluted with 25 mM aceticacid pH 4.5, and the pH was adjusted to pH 4.58. The pooled sample (100ml) was applied to a 1 ml HITRAP® SP Fast Flow column (GE Healthcare,Piscataway, N.J., USA) equilibrated with 25 mM acetic acid pH 4.5. Thebound protein was eluted with one column volume gradient from 0 to 500mM sodium chloride in 25 mM acetic acid pH 4.5. Based on SDS-PAGEanalysis and A280 and A₂₆₀ fractions were pooled (3.5 ml). The MW of thepurified xylanase was 20-25 kDa based on SDS-PAGE analysis.

Example 29: Preparation of Trichoderma reesei RutC30 GH3 Beta-Xylosidase

A Trichoderma reesei RutC30 beta-xylosidase gene (SEQ ID NO: 57 [DNAsequence] and SEQ ID NO: 58 [deduced amino acid sequence]) was isolatedby screening a Lambda ZAP®-CMR XR Library prepared from T. reesei RutC30genomic DNA using a Lambda ZAP®-CMR XR Library Construction Kit(Stratagene, La Jolla, Calif., USA) according to the manufacturer'sinstructions. T. reesei RutC30 genomic DNA was prepared using standardmethods. A DNA segment encoding 2300 bp of the T. reesei beta-xylosidasewas amplified using the PCR primers shown below.

Forward Primer: (SEQ ID NO: 149) 5'-gtgaataacgcagctcttctcg-3'Reverse Primer: (SEQ ID NO: 150) 5'-ccttaattaattatgcgtcaggtgt-3'Primer 994768 was designed to amplify from the first base after thebeta-xylosidase start site and primer 994769 was designed with a Pac Isite at the 5′ end.

Fifty picomoles of each of the primers above were used in a PCR reactionconsisting of 50 ng of plasmid DNA from the lamda zap library, 1 μl of10 mM blend of dATP, dTTP, dGTP, and dCTP, 5 μl of 10× PLATINUM® Pfx DNAPolymerase Buffer, and 1 unit of PLATINUM® Pfx DNA Polymerase, in afinal volume of 50 μl. An EPPENDORF® MASTERCYCLER® 5333 was used toamplify the DNA fragment programmed for one cycle at 95° C. for 3minutes; and 30 cycles each at 94° C. for 45 seconds, 55° C. for 60seconds, and 72° C. for 1 minute 30 seconds. After the 30 cycles, thereaction was incubated at 72° C. for 10 minutes and then cooled to 4° C.until further processing.

A 2.3 kb PCR product was purified by 1% agarose gel electrophoresisusing TAE buffer, excised from the gel, and purified using a QIAQUICK®Gel Extraction Kit. The 2.3 kb PCR product was then digested with Pac Ito facilitate insertion into pAILo1 (WO 2004/099228).

The pAILo1 vector was digested with Nco I and then filled in using T4polymerase (Roche, Nutley, N.J., USA) according to manufacturer'sinstructions. A second enzyme, Pac I, was then used to digest the 5′ endof pAILo1 and the reaction was purified by agarose gel electrophoresisas described above to isolate a 6.9 kb vector fragment.

The 2.3 kb beta-xylosidase fragment was then ligated into the 6.9 kbvector fragment and transformed into E. coli XL1-Blue SubcloningCompetent Cells (Invitrogen, Carlsbad, Calif., USA) according tomanufacturer's instructions. Transformants were screened usingrestriction digest analysis in order to identify those with the correctinsert. A new expression vector, pSaMe04, was confirmed by sequencingusing an AB13700 (Applied Biosystems, Foster City, Calif.) and dyeterminator chemistry (Giesecke et al., 1992, Journal of Virology Methods38: 47-60).

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Trichoderma reesei beta-xylosidase gene from pSaMe04 toconstruct a Trichoderma expression vector. An IN-FUSION™ Cloning Kit wasused to clone the fragment directly into the expression vector pMJ09 (WO2005/056772), without the need for restriction digestion and ligation.

TrBXYL-F (ID 064491): (SEQ ID NO: 151)5′-CGGACTGCGCACCATGGTGAATAACGCAGCTCT-3′ TrBXYL-R (ID 064492):(SEQ ID NO: 152) 5′-TCGCCACGGAGCTTATTATGCGTCAGGTGTAGCAT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pMJ09.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 50 ng of pSaMe04, 1 μl of 10 mM blend of dATP, dTTP, dGTP,and dCTP, 5 μl of 10× ACCUTAQ™ DNA Polymerase Buffer (Sigma-Aldrich, St.Louis, Mo., USA), and 5 units of ACCUTAQ™ DNA Polymerase (Sigma-Aldrich,St. Louis, Mo., USA), in a final volume of 50 μl. An EPPENDORF®MASTERCYCLER® 5333 was used to amplify the DNA fragment programmed forone cycle at 95° C. for 3 minutes; and 30 cycles each at 94° C. for 45seconds, 55° C. for 60 seconds, and 72° C. for 1 minute 30 seconds.After the 30 cycles, the reaction was incubated at 72° C. for 10 minutesand then cooled to 4° C. until further processing.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1.2 kb product band was excised from the geland purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The 1.2 kb fragment was then cloned into pMJ09 using an IN-FUSION™Cloning Kit. The vector was digested with Nco I and Pac I and purifiedby agarose gel electrophoresis as described above. The gene fragment andthe digested vector were ligated together in a reaction resulting in theexpression plasmid pSaMe-TrBXYL in which transcription of thebeta-xylosidase gene was under the control of the T. reesei cbh1 genepromoter. The ligation reaction (50 μl) was composed of 1× IN-FUSION™Buffer, 1×BSA, 1 μl of IN-FUSION™ enzyme (diluted 1:10), 100 ng of pMJ09digested with Nco I and Pac I, and 100 ng of the Trichoderma reeseibeta-xylosidase purified PCR product. The reaction was incubated at roomtemperature for 30 minutes. One μl of the reaction was used to transformE. coli XL10 SOLOPACK® Gold cells. An E. coli transformant containingpSaMe-TrBXYL was detected by restriction enzyme digestion and plasmidDNA was prepared using a BIOROBOT® 9600. DNA sequencing of theTrichoderma reesei beta-xylosidase gene from pSaMe-TrBXYL was performedusing dye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy.

Plasmid pSaMe-AaXYL was constructed to comprise the Trichoderma reeseicellobiohydrolase I gene promoter and terminator and the Aspergillusaculeatus GH10 xylanase coding sequence.

Cloning of the Aspergillus aculeatus xylanase followed the overallexpression cloning protocol as outlined in H. Dalboge et al., 1994, Mol.Gen. Genet. 243: 253-260.

RNA was isolated from Aspergillus aculeatus CBS 101.43 mycelium.Poly(A)⁺ RNA was isolated from total RNA by chromatography onoligo(dT)-cellulose. Double-stranded cDNA was synthesized as describedby Maniatis et al. (Molecular cloning: a laboratory manual. Cold SpringHarbor Laboratory Press, 1982). After synthesis the cDNA was treatedwith mung bean nuclease, blunt-ended with T4 DNA polymerase, and ligatedto non-palindromic Bst XI adaptors (Invitrogen, Carlsbad, Calif., USA).The cDNA was size fractionated by 1% agarose gel electrophoresis usingTAE buffer where fragments ranging from 600 bp to 4000 bp were used inthe library construction. The DNA was ligated into Bst XI-digested pYES2.0 between the GAL1 promoter and the iso-1-cytochrome c terminator andtransformed into Escherichia coli MC1061 cells (Stratagene, La Jolla,Calif., USA. The library was plated onto LB plates and incubatedovernight at 37° C. The colonies were scraped from the plates andresuspended in LB medium supplemented with 100 μg of ampicillin per ml.Plasmid DNA was isolated using a Plasmid Midi Kit (QIAGEN Inc.,Valenicia, Calif., USA). The purified plasmid DNA was pooled.

The purified plasmid DNA mixture was transformed into Saccharomycescerevisiae W3124 cells (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep4-1137; prcl::HIS3; prbl:: LEU2; cir+; van den Hazel et al., 1992, Eur.J. Biochem. 207: 277-283). Cultivation, transformation and media were asdescribed by Guthrie et al., 1991, Meth. Enzymol. Vol 194, AcademicPress. The transformed cells were plated onto synthetic complete agarcontaining 2% glucose for 3 days at 30° C. After 3 days the colonieswere replica plated to SC medium with 2% galactose and incubated for 4days at 30° C. Xylanase expressing colonies were identified by 1%agarose overlay with 0.1% AZCL-Birch-Xylan at pH 4.5 (Dalboge, 2006,FEMS Microbiology Reviews 21: 29-42). Colonies expressing xylanaseactivity were surrounded by a blue zone. Plasmid DNA, rescued from thepositive colonies, contained a DNA insert of approximately 1.3 kb.Sequencing of the isolated gene fragment revealed a 1218 bp open readingframe encoding a polypeptide with a theoretical molecular weight of 43.0kDa. The cDNA fragment was subcloned into the Aspergillus expressionvector pHD464 (Dalbøge and Heldt-Hansen, 1994, Mol. Gen. Genet. 243,253-260) digested with Barn HI and Xho i by cutting the done with BarnHI and Xho I and isolating the 1.2 kb cDNA insert (Christgau et al.,1996, Biochem. J. 319: 705-712) to generate plasmid pA2X2.

The Aspergillus aculeatus GH10 xylanase coding sequence was PCRamplified using plasmid pA2x2 as template and primers 153505 and 153506shown below using standard methods to yield an approximately 1.2 kbfragment. The 1.2 kb fragment was digested with Barn HI and Xho I(introduced in the PCR primers) and cloned into vector pCaHj527 (WO to2004/099228). The resulting plasmid was designated pMT2155 in which thecDNA was under transcriptional control of the neutral amylase II (NA2)promoter from A. niger and the AMG terminator from A. niger.

Primer 153505: (SEQ ID NO: 153)5′-TCTTGGATCCACCATGGTCGGACTGCTTTCAATCACC-3′ Primer 153506:(SEQ ID NO: 154) 5′-TTAACTCGAGTCACAGACACTGCGAGTAATAGTC-3′

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Aspergillus aculeatus GH10 gene from plasmid pMT2155 andintroduce flanking regions for insertion into expression vector pMJ09(WO 2005/056772). Bold letters represent coding sequence and theremaining sequence is homologous to the insertion sites of pMJ09.

Forward Primer: (SEQ ID NO: 155) 5′-cggactgcgcaccatggtcggactgctttcaat-3′Reverse Primer: (SEQ ID NO: 156)5′-tcgccacggagcttatcacagacactgcgagtaat-3′

Fifty picomoles of each of the primers above were used in a PCR reactionconsisting of 50 ng of pMT2155, 1 μl of 10 mM blend of dATP, dTTP, dGTP,and dCTP, 5 μl of 10× ACCUTAQ™ DNA Polymerase Buffer, and 5 units ofACCUTAQ™ DNA Polymerase, in a final volume of 50 μl. An EPPENDORF®MASTERCYCLER® 5333 was used to amplify the DNA fragment programmed forone cycle at 95° C. for 3 minutes; and 30 cycles each at 94° C. for 45seconds, 55° C. for 60 seconds, and 72° C. for 1 minute 30 seconds.After the 30 cycles, the reaction was incubated at 72° C. for 10 minutesand then cooled to 4° C. until further processing.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer where a 1.2 kb product band was excised from the gel and purifiedusing a QlAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The fragment was then cloned into pMJ09 using an IN-FUSION™ Cloning Kit.The vector was digested with Nco I and Pac I and purified by agarose gelelectrophoresis as described above. The 1.2 kb gene fragment and thedigested vector were ligated together in a reaction resulting in theexpression plasmid pSaMe-AaXYL in which transcription of the Family GH10gene was under the control of the T. reesei cbh1 promoter. The ligationreaction (50 ul) was composed of 1× IN-FUSION™ Buffer, 1×BSA, 1 μl ofIN-FUSION™ enzyme (diluted 1:10), 100 ng of pAILo2 digested with Nco Iand Pac I, and 100 ng of the Aspergillus aculeatus GH10 xylanasepurified PCR product. The reaction was incubated at room temperature for30 minutes. One μl of the reaction was used to transform E. coli XL10SOLOPACK® Gold cells according to the manufacturer. An E. colitransformant containing pSaMe-AaGH10 was detected by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600. DNAsequencing of the Aspergillus aculeatus GH10 gene from pSaMe-AaXYL wasperformed using dye-terminator chemistry (Giesecke et al., 1992, supra)and primer walking strategy.

Plasmids pSaMe-AaXYL encoding the Aspergillus aculeatus GH10endoglucanase and pSaMe-TrBXYL encoding the Trichoderma reeseibeta-xylosidase were co-transformed into Trichoderma reesei RutC30 byPEG-mediated transformation (Penttila et al., 1987, Gene 61 155-164) togenerate T. reesei strain SaMe-BXX13. Each plasmid contained theAspergillus nidulans amdS gene to enable transformants to grow onacetamide as the sole nitrogen source.

Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17hours. Mycelia were collected by filtration using a Vacuum DrivenDisposable Filtration System (Millipore, Bedford, Mass., USA) and washedtwice with deionized water and twice with 1.2 M sorbitol. Protoplastswere generated by suspending the washed mycelia in 20 ml of 1.2 Msorbitol containing 15 mg of GLUCANEX™ (Novozymes A/S, Bagsvaerd,Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St.Louis, Mo., USA) per ml and incubating for 15-25 minutes at 34° C. withgentle shaking at 90 rpm. Protoplasts were collected by centrifuging for7 minutes at 400×g and washed twice with cold 1.2 M sorbitol. Theprotoplasts were counted using a haemacytometer and re-suspended in STCto a final concentration of 1×10⁸ protoplasts per ml. Excess protoplastswere stored in a Cryo 1° C. Freezing Container (Nalgene, Rochester,N.Y., USA) at −80° C.

Approximately 4 μg of plasmids pSaMe-AaXYL and pSaMe-TRBXYL weredigested with Pme I and added to 100 μl of protoplast solution and mixedgently, followed by 250 μl of 10 mM CaCl₂-10 mM Tris-HCl pH 7.5-60% PEG4000, mixed, and incubated at room temperature for 30 minutes. STC (3ml) was then added and mixed and the transformation solution was platedonto COVE plates using Aspergillus nidulans amdS selection. The plateswere incubated at 28° C. for 5-7 days. Transformants were sub-culturedonto COVE2 plates and grown at 28° C.

Over 40 transformants were subcultured onto fresh plates containingacetamide and allowed to sporulate for 7 days at 28° C.

The Trichoderma reesei transformants were cultivated in 125 ml baffledshake flasks containing 25 ml of cellulase-inducing medium at pH 6.0 byinoculating spores of the transformants and incubating at 28° C. and 200rpm for 7 days. Trichoderma reesei RutC30 was run as a control. Culturebroth samples were removed at day 5. One ml of each culture broth wascentrifuged at 15,700×g for 5 minutes in a micro-centrifuge and thesupernatants transferred to new tubes.

SDS-PAGE was performed using CRITERION® Tris-HCl (5% resolving) gels(Bio-Rad Laboratories, Inc.) with a CRITERION® System. Five μl of day 7supernatants (see above) were suspended in 2× concentration of LaemmliSample Buffer (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) andboiled in the presence of 5% beta-mercaptoethanol for 3 minutes. Thesupernatant samples were loaded onto a polyacrylamide gel and subjectedto electrophoresis with 1×Tris/Glycine/SDS as running buffer (Bio-RadLaboratories, Inc., Hercules, Calif., USA). The resulting gel wasstained with BIO-SAFE™ Coomassie Stain. The transformant showing thehighest expression of both the A. aculeatus GH10 xylanase and the T.reesei beta-xylosidase based on the protein gel was designated T. reeseiSaMe-BXX13.

Trichoderma reesei SaMe-BXX13 was cultivated in 500 ml baffled shakeflasks containing 250 ml of cellulase-inducing medium at pH 6.0inoculated with spores of T. reesei SaMe-BXX13. Shake flasks wereincubated at 28° C. at 200 rpm for five days. The culture broth was thenfiltered using an 0.22 μm EXPRESS™ Plus Membrane.

The filtered broth was concentrated and buffer exchanged using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane to pH 4.0 with acetic acid. Sample was loaded onto a SPSEPHAROSE® column equilibrated in 50 mM sodium acetate pH 4.0, elutingbound proteins with a gradient of 0-1000 mM sodium chloride. Fractionswere buffer exchanged into 20 mM sodium phosphate pH 7.0 using atangential flow concentrator and applied to a Phenyl SUPEROSE™ column(HR 16/10) equilibrated with 1.5 M (NH₄)₂SO₄-20 mM sodium phosphate pH7.0. Bound proteins were eluted with a linear gradient over 20 columnvolumes from 1.5 to 0 M (NH₄)₂SO₄ in 20 mM Tris-HCl pH 7.0. The proteinfractions were buffer exchanged into 20 mM TEA HCl pH 7.5 using atangential flow concentrator. Sample was applied to a MonoQ® column,equilibrated in 20 mM TEA HCl pH 7.5, eluting bound proteins with agradient from 0-300 mM sodium chloride. Buffer of final proteinfractions was 20 mM TEA-100 mM sodium chloride pH 7.5. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 30: Preparation of Talaromyces emersonii CBS 393.64 GH3Beta-Xylosidase

Talaromyces emersonii CBS 393.64 (NN005049) beta-xylosidase (SEQ ID NO:59 [DNA sequence] and SEQ ID NO: 60 [deduced amino acid sequence]) wasprepared recombinantly according to Rasmussen et al., 2006,Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzaeJaL355 as a host (WO 2003/070956).

The Talaromyces emersonii beta-xylosidase was purified according toRasmussen et al., 2006, supra.

Example 31: Preparation of Trichoderma reesei RutC30 Cel7B EndoglucanaseI

Trichoderma reesei RutC30 Cel7B endoglucanase I (EGI) (SEQ ID NO: 61[DNA sequence] and SEQ ID NO: 62 [deduced amino acid sequence]) wasprepared recombinantly according to WO 2005/067531 using Aspergillusoryzae JaL250 as a host.

The harvested broth was centrifuged in 500 ml bottles at 13,000×g for 20minutes at 4° C. and then sterile filtered using a 0.22 μmpolyethersulfone membrane (Millipore, Bedford, Mass., USA). The filteredbroth was concentrated and buffer exchanged with 20 mM Tris-HCl pH 8.5using a tangential flow concentrator equipped with a 10 kDapolyethersulfone membrane. The sample was loaded onto a Q SEPHAROSE®High Performance column equilibrated with 20 mM Tris-HCl pH 8.5, andstep eluted with equilibration buffer containing 600 mM NaCl.Flow-through and eluate fractions were analyzed by SGS-PAGE gel analysisusing a CRITERION™ stain-free imaging system (Bio-Rad Laboratories,Inc., Hercules, Calif., USA). The eluate fractions containingTrichoderma reesei Cel7B EGI were pooled, concentrated and bufferexchanged into 20 mM Tris-HCl pH 8.5. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 32: Preparation of Trichoderma reesei RutC30 Cel7ACellobiohydrolase I

Trichoderma reesei RutC30 Cel7A cellobiohydrolase I (CBHI) (SEQ ID NO:63 [DNA sequence] and SEQ ID NO: 64 [deduced amino acid sequence]) wasprepared as described by Ding and Xu, 2004, “Productive cellulaseadsorption on cellulose” in Lignocellulose Biodegradation (Saha, B. C.ed.), Symposium Series 889, pp. 154-169, American Chemical Society,Washington, D.C. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 33: Preparation of Trichoderma reesei RutC30 Cel6ACellobiohydrolase II

The Trichoderma reesei RutC30 Cel6A cellobiohydrolase II gene (SEQ IDNO: 65 [DNA sequence] and SEQ ID NO: 66 [deduced amino acid sequence])was isolated from Trichoderma reesei RutC30 as described in WO2005/056772.

The Trichoderma reesei Cel6A cellobiohydrolase II gene was expressed inFusarium venenatum using pEJG61 as an expression vector according to theprocedures described in U.S. Published Application No. 20060156437.Fermentation was performed as described in U.S. Published ApplicationNo. 20060156437.

Filtered broth was desalted and buffer-exchanged into 20 mM sodiumacetate-150 mM NaCl pH 5.0 using a HIPREP® 26/10 Desalting Columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 34: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. The PCS was used unwashed or washed with water.Milled unwashed PCS (dry weight 32.35%) was prepared by milling wholeslurry PCS in a Cosmos ICMG 40 wet multi-utility grinder (EssEmmCorporation, Tamil Nadu, India). Milled washed PCS (dry weight 32.35%)was prepared in the same manner, with subsequent washing with deionizedwater and decanting off the supernatant fraction repeatedly.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of PCS (insoluble solids in caseof unwashed PCS and total solids in case of washed PCS) per ml of 50 mMsodium acetate pH 5.0 buffer containing 1 mM manganese sulfate andvarious protein loadings of various enzyme compositions (expressed as mgprotein per gram of cellulose). Enzyme compositions were prepared andthen added simultaneously to all wells in a volume ranging from 50 μl to200 μl, for a final volume of 1 ml in each reaction. The plate was thensealed using an ALPS-300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at a specific temperature for72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.In case of unwashed PCS, the net concentrations ofenzymatically-produced sugars were determined by adjusting the measuredsugar concentrations for corresponding background sugar concentrationsin unwashed PCS at zero time point. All HPLC data processing wasperformed using MICROSOFT EXCEL™ software (Microsoft, Richland, Wash.,USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation:% conversion=glucose concentration/glucoseconcentration in a limit digest. To calculate total conversion theglucose and cellobiose values were combined. Cellobiose concentrationwas multiplied by 1.053 in order to convert to glucose equivalents andadded to the glucose concentration. The degree of total celluloseconversion was calculated using the following equation:

% conversion=[glucose concentration+1.053×(cellobioseconcentration)]/[(glucose concentration+1.053×(cellobiose concentration)in a limit digest].

The 1.053 factor for cellobiose takes into account the increase in masswhen cellobiose is converted to glucose. In order to calculate %conversion, a 100% conversion point was set based on a cellulase control(50-100 mg of Trichoderma reesei cellulase per gram cellulose), and allvalues were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 35: Evaluation of Several Cellulolytic Proteins Replacing CBHI,CBHII, and EGII Components in a Reconstituted Trichoderma reesei-BasedEnzyme Composition for Improved Performance at 50° C., 55° C., and 60°C.

Several cellulolytic proteins were tested in various combinations at 50°C., 55° C., and 60° C. against a reconstituted Trichoderma reesei-basedenzyme composition that included four major Trichoderma reeseicellulases (45% Trichoderma reesei Cel7A CBHI, 25% Trichoderma reeseiCel6A CBHII, 5% Trichoderma reesei Cel7B EGI, 5% Trichoderma reeseiCel5A EGII), a beta-glucosidase (10% Aspergillus fumigatus Cel3Abeta-glucosidase), and a Family 61 polypeptide having cellulolyticenhancing activity (10% Thermoascus aurantiacus GH61A polypeptide).

The evaluated enzymes included Chaetomium thermophilum Cel7A CBHI,Myceliophthora thermophila Cel7A CBHI, Myceliophthora thermophila Cel6ACBHII, and Myceliophthora thermophila Cel5A EGII. All enzymecompositions contained 45% CBHI, 25% CBHII, 5% Trichoderma reesei Cel7BEGI, 5% EGII, 10% Aspergillus fumigatus beta-glucosidase, and 10%Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity. All enzyme compositions, including the Trichodermareesei-based composition, were applied at the same dosage of 5 mgprotein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results as shown in FIG. 1 demonstrated that the best enzymecomposition for cellulose hydrolysis included Chaetomium thermophilumCel7A CBHI, Myceliophthora thermophila Cel6A CBHII, Trichoderma reeseiCel7B EGI, Myceliophthora thermophila Cel5A EGII, Aspergillus fumigatusbeta-glucosidase, and Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity. Replacement of the Trichoderma reeseiCel7A CBHI, Cel6A CBHII, and Cel5A EGII with Chaetomium thermophilumCel7A CBHI, Myceliophthora thermophila Cel6A CBHII, and Myceliophthorathermophila Cel5A EGII significantly improved the degree of celluloseconversion to glucose at 60° C. (from 50% to 65%). The improvedcomposition hydrolyzed milled washed PCS almost as efficiently at 60° C.(65% cellulose conversion to glucose) as the Trichoderma reesei—basedcomposition at 50° C. (68%).

Example 36: Evaluation of GH61 Polypeptides Having CellulolyticEnhancing Activity for the Ability to Enhance PCS-Hydrolyzing Activityof a High-Temperature Enzyme Composition at 50° C., 55° C., and 60° C.

Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity and Thielavia terrestris GH61E polypeptide having cellulolyticenhancing activity, prepared as described herein, were tested at 10%total protein addition for the ability to stimulate a high-temperaturecellulase composition at 50° C., 55° C., and 60° C. using milled washedPCS. The compositions consisting of 45% Chaetomium thermophilum Cel7ACBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5% Trichoderma reeseiCel7B EGI, 5% Myceliophthora thermophila Cel5A EGII, 10% Aspergillusfumigatus beta-glucosidase, and 10% GH61 polypeptide having cellulolyticenhancing activity were used for hydrolysis of milled washed PCS at 5 mgprotein per g cellulose, and the results were compared with the resultsfor a high-temperature enzyme composition without a GH61 polypeptide,which was used at 4.5 mg protein per g cellulose. The assay wasperformed as described in Example 34. The 1 ml reactions with 5% milledwashed PCS were conducted for 72 hours in 50 mM sodium acetate pH 5.0buffer containing 1 mM manganese sulfate. All reactions were performedin triplicate and involved single mixing at the beginning of hydrolysis.

The results shown in FIG. 2 demonstrated that the Thermoascusaurantiacus GH61A and Thielavia terrestris GH61E polypeptides were ableto significanty enhance PCS-hydrolyzing activity of the high-temperatureenzyme composition at all temperatures from 50° C. to 60° C., and thatthe stimulating effect was more pronounced at higher temperatures. At60° C., a composition without a GH61 polypeptide showed 48% conversionof cellulose to glucose. For comparison, compositions that includedThermoascus aurantiacus GH61A or Thielavia terrestris GH61E polypeptidesshowed 70% and 68% conversion of cellulose to glucose, respectively.

Example 37: Evaluation of a Binary Composition of Thermoascusaurantiacus GH61A and Thielavia terrestris GH61E GH61 PolypeptidesHaving Cellulolytic Enhancing Activity for the Ability to EnhancePCS-Hydrolyzing Activity of a High-Temperature Enzyme Composition at 50°C., 55° C., and 60° C.

The boosting performance of a binary composition comprising equalamounts (on a protein basis) of Thermoascus aurantiacus GH61A andThielavia terrestris GH61E GH61 polypeptides having cellulolyticenhancing activity was compared with the boosting performance of theindividual GH61 proteins alone at equivalent total protein loading at50° C., 55° C., and 60° C. The high-temperature enzyme compositionsincluded 45% Chaetomium thermophilum Cel7A CBHI, 25% Myceliophthorathermophila Cel6A CBHII, 5% Trichoderma reesei Cel7B EGI, 5%Myceliophthora thermophila Cel5A EGII, 10% Aspergillus fumigatusbeta-glucosidase, and 10% GH61 component (either an individualpolypeptide or a binary composition). The total protein loading inhydrolysis reactions was 5 mg protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results shown in FIG. 3 demonstrated that the binary combination ofthe two GH61 polypeptides having cellulolytic enhancing activity,Thermoascus aurantiacus GH61A and Thielavia terrestris GH61E, providedgreater enhancement than either of the GH61 proteins alone. The effectwas especially pronounced at 60° C.

Example 38: Evaluation of Binary Compositions Containing DifferentRatios of Thermoascus aurantiacus GH61A and Thielavia terrestris GH61EGH61 Polypeptides Having Cellulolytic Enhancing Activity for the Abilityto Enhance PCS-Hydrolyzing Activity of a High-Temperature EnzymeComposition at 60° C.

After determining that a 1:1 composition of Thermoascus aurantiacusGH61A and Thielavia terrestris GH61E GH61 polypeptides havingcellulolytic enhancing activity performed better in enhancing thePCS-hydrolyzing activity of a high-temperature enzyme composition thaneither protein alone, the effect was analyzed in more detail byexamining different ratios of the Thermoascus aurantiacus GH61A andThielavia terrestris GH61E polypeptides. The various binary compositionsof the GH61 polypeptides were added at 0.5 mg protein per g cellulose toa high-temperature enzyme composition (4.5 mg protein per g cellulose)so that the final mixture consisted of 45% Chaetomium thermophilum Cel7ACBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5% Trichoderma reeseiCel7B EGI, 5% Myceliophthora thermophila Cel5A EGII, 10% Penicilliumbrasilianum beta-glucosidase, and 10% GH61 component (either anindividual GH61 polypeptide or a binary composition).

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results are shown in Table 1 and FIG. 4. All binary compositions ofThermoascus aurantiacus GH61A and Thielavia terrestris GH61E GH61polypeptides having cellulolytic enhancing activity providedsignificantly better enhancement of PCS hydrolysis by thehigh-temperature enzyme composition than either protein alone. The bestperformance was obtained for binary GH61 compositions that included atleast 20% of either Thermoascus aurantiacus GH61A or Thielaviaterrestris GH61E polypeptide having cellulolytic enhancing activity.

TABLE 1 Addition of binary GH61 polypeptide compositions (0.5 mg proteinper g cellulose) to a high-temperature enzyme composition (4.5 mgprotein per g cellulose) at 60° C. mg protein per g cellulose % of totalGH61 addition Thermoascus Thiela via Thermoascus Thielavia % aurantiacusterrestris aurantiacus terrestris Con- GH61A GH61E GH61A GH61E version0.00 0.00 0 0 47.9 0.00 0.50 0 100 71.0 0.05 0.45 10 90 79.0 0.10 0.4020 80 81.3 0.15 0.35 30 70 81.8 0.20 0.30 40 60 82.0 0.25 0.25 50 5082.7 0.30 0.20 60 40 81.8 0.35 0.15 70 30 81.2 0.40 0.10 80 20 80.6 0.450.05 90 10 78.8 0.50 0.00 100 0 72.8

Example 39: Evaluation of Different Levels of a Binary 1:1 CompositionComprising Thermoascus aurantiacus GH61A and Thielavia terrestris GH61EGH61 Polypeptides Having Cellulolytic Enhancing Activity for the Abilityto Enhance PCS-Hydrolyzing Activity of a High-Temperature EnzymeComposition at 60° C.

The ability of Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, Thielavia terrestris GH61E polypeptidehaving cellulolytic enhancing activity, or their binary 1:1 composition(on a protein basis) to stimulate a high-temperature enzyme compositionwas examined by adding different GH61 protein loadings (0.125, 0.25,0.5, 1.0, 1.5 mg protein per g cellulose) to a constant loading of thehigh-temperature enzyme composition (4.5 mg per g cellulose) at 60° C.The high-temperature enzyme composition contained 45% Chaetomiumthermophilum Cel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5%Trichoderma reesei Cel7B EGI, 5% Myceliophthora thermophila Cel5A EGII,and 10% Penicillium brasilianum Cel3A beta-glucosidase.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results shown in FIG. 5 demonstrated that at equivalent proteinloadings, the 1:1 compositions of Thermoascus aurantiacus GH61A andThielavia terrestris GH61E GH61 polypeptides having cellulolyticenhancing activity provided greater PCS hydrolysis enhancement thaneither of the GH61 proteins alone. The effect was especially pronouncedat relatively high additions of the GH61 proteins. The effect saturatedwhen the level of the GH61 binary composition reached approximately 20%of the total protein loading.

Example 40: Effect of Thermobifida fusca GH11 Xylanase onSaccharification of Milled Washed PCS by a High-Temperature EnzymeComposition at 50-65° C.

The ability of Thermobifida fusca GH11 xylanase (0.5 mg protein per gcellulose) to stimulate saccharification of milled washed PCS by ahigh-temperature enzyme composition (5 mg protein per g cellulose) wasexamined at 50° C., 55° C., 60° C., and 65° C. For comparison, thehigh-temperature enzyme composition without a supplemental xylanase wastested at 5.0, 5.5, and 6.0 mg protein per g cellulose. Thehigh-temperature enzyme composition included 45% Chaetomium thermophilumCel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5% Trichodermareesei Cel7B EGI, 5% Myceliophthora thermophila Cel5A EGII, 10%Penicillium brasilianum Cel3A beta-glucosidase, and 10% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results shown in FIG. 6 demonstrated that addition of Thermobifidafusca GH11 xylanase significantly improved performance of thehigh-temperature enzyme composition at all temperatures from 50° C. to65° C., increasing the cellulose conversion to glucose by 4-7% after 72hours of hydrolysis.

Example 41: Replacement of Chaetomium thermophilum Cel7ACellobiohydrolase I in a High-Temperature Enzyme Composition withVarious Thermostable CBHI Proteins at 50-65° C.

The ability of several thermostable CBHI proteins to replace Chaetomiumthermophilum Cel7A CBHI in a high-temperature enzyme composition (4 mgtotal protein per g cellulose) was tested at 50° C., 55° C., 60° C., and65° C. The high-temperature enzyme composition included 45% Chaetomiumthermophilum Cel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 10%Myceliophthora thermophila Cel5A EGII, 5% Thermoascus aurantiacus GH61Apolypeptide having cellulolytic enhancing activity, 5% Thielaviaterrestris GH61E polypeptide having cellulolytic enhancing activity, and10% Penicillium brasilianum Cel3A beta-glucosidase.

The following recombinant CBHI cellulases from an Aspergillus oryzaeexpression host were tested as a replacement for Chaetomium thermophilumCel7A CBHI: Myceliophthora thermophila Cel7A, Aspergillus fumigatusCel7A, and Thermoascus aurantiacus Cel7A.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

As shown in FIG. 7, the replacement of Chaetomium thermophilum Cel7ACBHI with Aspergillus fumigatus Cel7A CBHI gave the best hydrolysisresults, providing an almost 6% improvement of the final hydrolysisyield at 60° C. Surprisingly, a native Thermoascus aurantiacus Cel7ACBHI, which contained no CBM, also showed a remarkably good performanceat elevated temperatures.

Example 42: Comparison of High-Temperature Enzyme CompositionsContaining Aspergillus fumigatus Cel7A CBHI or Chaetomium thermophilumCel7A CBHI with Trichoderma reesei-Based Cellulase SaMe-MF268 (XCL-533)at 50° C. and 60° C.

Two high-temperature enzyme compositions that included eitherAspergillus fumigatus Cel7A CBHI or Chaetomium thermophilum Cel7A CBHIwere tested in comparison with Trichoderma reesei-based cellulasecomposition SaMe-MF268 (XCL-533) at four protein loadings (3.5, 4.0,4.5, and 5.0 mg protein per g cellulose) and two temperatures (50° C.and 60° C.) using milled washed PCS as a substrate The high-temperatureenzyme compositions included 45% Cel7A CBHI, 25% Myceliophthorathermophila Cel6A CBHII, 5% Trichoderma reesei Cel7B EGI, 5%Myceliophthora thermophila Cel5A EGII, 5% Thermoascus aurantiacus GH61Apolypeptide having cellulolytic enhancing activity, 5% Thielaviaterrestris GH61E polypeptide having cellulolytic enhancing activity,7.5% Penicillium brasilianum GH3A beta-glucosidase, and 2.5%Thermobifida fusca GH11 xylanase. The Trichoderma reesei-based enzymecomposition SaMe-MF268 (XCL-533) was obtained as described in WO2008/151079. The composition comprises a Thermoascus aurantiacus GH61Apolypeptide having cellulolytic enhancing activity, a beta-glucosidasefusion protein comprising the Humicola insolens endoglucanase V corepolypeptide fused to the wild-type Aspergillus oryzae beta-glucosidase,a Trichoderma reesei Cel7A cellobiohydrolase I, a Trichoderma reeseiCel6A cellobiohydrolase II, a Trichoderma reesei Cel7B endoglucanase I,a Trichoderma reesei Cel5A endoglucanase II, a Trichoderma reesei Cel45Aendoglucanase V, and a Trichoderma reesei Cel12A endoglucanase III.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

Protein dose profiles for the Aspergillus fumigatus Cel7A CBHI-basedcomposition in comparison with the Chaetomium thermophilum Cel7ACBHI-based composition and the Trichoderma reesei-based cellulasecomposition XCL-533 are shown in FIG. 8. The Trichoderma reesei-basedcellulase composition XCL-533 showed poor performance at 60° C., whileboth high-temperature enzyme compositions were significantly activatedby the temperature increase from 50° C. to 60° C. The high-temperatureenzyme composition that included Aspergillus fumigatus Cel7A CBHIperformed at 60° C. as well as the Trichoderma reesei-based cellulasecomposition XCL-533 performed at its optimum temperature of 50° C.,requiring approximately 3.5 mg protein per g cellulose to achieve 80%conversion of cellulose to glucose in 72 hours. For the Chaetomiumthermophilum Cel7A CBHI-based composition, the protein loading requiredto achieve the same degree of cellulose conversion at 60° C. was 4.5 mgprotein per g cellulose.

Example 43: Hydrolysis Time-Course for Aspergillus fumigatus Cel7ACBHI-Based High-Temperature Enzyme Composition in Comparison withTrichoderma reesei-Based Cellulase Composition SaMe-MF268 at 50° C. and60° C.

Hydrolysis performance of the Aspergillus fumigatus Cel7A CBHI-basedhigh-temperature enzyme composition and the Trichoderma reesei-basedcellulase composition SaMe-MF268 (XCL-533) was compared over a longerincubation time (five days) at 50° C. and 60° C. using milled washed PCSas a substrate. The Aspergillus fumigatus Cel7A CBHI-based enzymecomposition included 45% Aspergillus fumigatus Cel7A CBHI, 25%Myceliophthora thermophila Cel6A CBHII, 5% Trichoderma reesei Cel7B EGI,5% Myceliophthora thermophila Cel5A EGII, 5% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Thielaviaterrestris GH61E polypeptide having cellulolytic enhancing activity,7.5% Penicillium brasilianum GH3A beta-glucosidase, and 2.5%Thermobifida fusca GH11 xylanase. The Aspergillus fumigatus Cel7ACBHI-based enzyme composition and the Trichoderma reesei-based cellulasecomposition XCL-533 were tested at four different protein loadings, 2.0,3.0, 3.5, and 4.0 mg protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The time-course hydrolysis results for one of the four tested proteinloadings (2 mg protein per g cellulose) are shown in FIG. 9. Similartrends were obtained for the three other protein loadings (data notshown). The Trichoderma reesei-based cellulase composition XCL-533showed significantly reduced performance at 60° C., while theAspergillus fumigatus Cel7A CBHI-based enzyme composition wassignificantly activated by the temperature increase from 50° C. to 60°C.

Comparison of the Aspergillus fumigatus Cel7A CBHI-based enzymecomposition at 60° C. and the Trichoderma reesei-based cellulasecomposition XCL-533 at 50° C. showed that the high-temperaturecomposition performed better than the Trichoderma reesei-based cellulasecomposition XCL-533 during the initial three days, and similarly to theTrichoderma reesei-based cellulase composition XCL-533 during the lasttwo days of hydrolysis. Comparison of the Aspergillus fumigatus Cel7ACBHI-based enzyme composition and the Trichoderma reesei-based cellulasecomposition XCL-533 at the same temperature of 50° C. showed slower butsteadier rates of glucose accumulation for the high-temperaturecomposition in comparison with the Trichoderma reesei-based cellulasecomposition XCL-533, resulting in a better performance of thehigh-temperature composition in a long-term hydrolysis at 50° C. (5days).

Example 44: Evaluation of Four Xylanases for Synergy with theAspergillus fumigatus Cel7A CBH I-Based High-Temperature EnzymeComposition at 50° C., 55° C., and 60° C.

Aspergillus aculeatus GH10 xylanase II, Aspergillus fumigatus GH10 xyn3xylanase, Trichophaea saccata GH10 xylanase, and Thermobifida fusca GH11xylanase were assayed for synergy with a high-temperature enzymecomposition containing Cel7A CBHI from Aspergillus fumigatus at 50° C.,55° C., and 60° C. using milled washed PCS as a substrate. The xylanaseswere added at 10% (0.35 mg protein per g cellulose) to a constantloading of the high-temperature enzyme composition (3.5 mg protein per gcellulose). The high-temperature enzyme composition included 45%Aspergillus fumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6ACBHII, 5% Trichoderma reesei Cel7B EGI, 5% Myceliophthora thermophilaCel5A EGII, 5% Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, 5% Thielavia terrestris GH61Epolypeptide having cellulolytic enhancing activity, and 10% Penicilliumbrasilianum Cel3A beta-glucosidase.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

The results shown in FIG. 10 demonstrated a considerable synergy betweenthe xylanases and the high-temperature enzyme composition. At anequivalent protein loading (3.85 mg protein per g cellulose), all enzymecompositions that included xylanases achieved a significantly higherdegree of cellulose conversion compared to the non-supplementedhigh-temperature enzyme composition. GH10 xylanases from Aspergillusfumigatus (xyn3) and Trichophaea saccata showed better performance thanAspergillus aculeatus GH10 xylanase II and Thermobifida fusca GH11xylanase. The addition of the top two xylanases to the high-temperatureenzyme composition resulted in an additional 11-16% conversion ofcellulose to glucose in 72 hours compared to the non-supplemented enzymecomposition (3.85 mg protein per g cellulose).

Example 45: Evaluation of Aspergillus fumigatus GH10 Xylanase Xyn3,Trichophaea Saccata GH10 Xylanase, and Thermobifida fusca GH11 Xylanasefor Synergy with a High-Temperature Enzyme Composition at 60° C.

The ability of Aspergillus fumigatus GH10 xyn3 xylanase, Trichophaeasaccata GH10 xylanase, and Thermobifida fusca GH11 xylanase to synergizewith a high-temperature enzyme composition containing Aspergillusfumigatus Cel7A CBHI was further examined by adding different levels ofeach xylanase (1.25%, 2.5%, 5%, 10%, and 20%) to a constant loading ofthe high-temperature enzyme composition (3 mg protein per g cellulose)at 60° C. using washed milled PCS as a substrate. The high-temperatureenzyme composition included 45% Aspergillus fumigatus Cel7A CBHI, 25%Myceliophthora thermophila Cel6A CBHII, 5% Trichoderma reesei Cel7B EGI,5% Myceliophthora thermophila Cel5A EGII, 5% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Thielaviaterrestris GH61E polypeptide having cellulolytic enhancing activity, and10% Penicillium brasilianum Cel3A beta-glucosidase.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis. The results are shown in FIG. 11.

Aspergillus fumigatus GH10 xyn3 and Trichophaea saccata GH10 xylanasesperformed similarly and showed better enhancement of PCS hydrolysis thanThermobifida fusca GH11 xylanase. The high-temperature enzymecomposition supplemented with the GH10 xylanase from Aspergillusfumigatus or Trichophaea saccata significantly outperformed theTrichoderma reesei-based cellulase composition SaMe-MF268 (XCL-533)after 72 hours of incubation with milled washed PCS. The optimaladdition level was about 5% for the Aspergillus fumigatus GH10 xyn3 andTrichophaea saccata GH10 xylanases and about 10% for the Thermobifidafusca GH11 xylanase. As shown in FIG. 11, the addition of eitherAspergillus fumigatus GH10 xyn3 or Trichophaea saccata GH10 xylanase tothe high-temperature composition at a level of only 5% at 60° C.enhanced the cellulose conversion to glucose from 65% to 83%. Anequivalent loading of the Trichoderma reesei-based cellulase compositionXCL-533 (3.15 mg protein per g cellulose) yielded 73% conversion ofcellulose to glucose at 50° C.

Example 46: Comparison of Aspergillus fumigatus Cel7A-BasedHigh-Temperature Enzyme Composition Containing Aspergillus fumigatusGH10 Xyn3 Xylanase with Trichoderma reesei-Based Cellulase SaMe-MF268

A high-temperature enzyme composition containing Aspergillus fumigatusGH10 xyn3 xylanase and the Trichoderma reesei-based cellulasecomposition SaMe-MF268 (XCL-533) were tested at three different proteinloadings, 2.0, 3.0, and 4.0 mg protein per g cellulose, and the proteinloading profile of the high-temperature enzyme composition at 60° C. wascompared with the protein loading profile of the Trichodermareesei-based cellulase composition XCL-533 at 50° C. Thehigh-temperature enzyme composition contained 45% Aspergillus fumigatusCel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5% Trichodermareesei Cel7B EGI, 5% Myceliophthora thermophila Cel5A EGII, 5%Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity, 5% Thielavia terrestris GH61E polypeptide having cellulolyticenhancing activity, 5% Penicillium brasilianum Cel3A beta-glucosidase,and 5% Aspergillus fumigatus GH10 xyn3.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled washed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis. The results are shown in FIG. 12.

The experiment confirmed that the improved high-temperature enzymecomposition containing Aspergillus fumigatus GH10 xyn3 xylanasesignificantly surpassed the performance of the Trichoderma reesei-basedcellulase composition XCL-533. At 60° C., the high-temperature enzymecomposition required a significantly lower protein loading (2.75 mgprotein per g cellulose) than the Trichoderma reesei-based cellulasecomposition XCL-533 at 50° C. (3.80 mg protein per g cellulose) tohydrolyze 80% of cellulose to glucose in 72 hours.

Example 47: Comparison of High-Temperature Enzyme CompositionsContaining Aspergillus fumigatus GH10 Xyn3 or Trichophaea saccata GH10Xylanase with Trichoderma reesei-Based Cellulase SaMe-MF268 inHydrolysis of Washed and Unwashed PCS

Protein loading profiles of the improved high-temperature enzymecompositions containing either GH10 xylanase from Aspergillus fumigatus(xyn 3) or GH10 xylanase from Trichophaea saccata were compared withprotein loading profiles of the Trichoderma reesei-based cellulasecomposition SaMe-MF268 (XCL-533) using milled washed and milled unwashedPCS. The high-temperature enzyme compositions included 45% Aspergillusfumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5%Trichoderma reesei Cel7B EGI, 5% Myceliophthora thermophila Cel5A EGII,5% Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity, 5% Thielavia terrestris GH61E polypeptide havingcellulolytic enhancing activity, 5% Penicillium brasilianum Cel3Abeta-glucosidase, and 5% GH10 xylanase. The high-temperature enzymecompositions and the Trichoderma reesei-based cellulase compositionXCL-533 were tested at five different protein loadings, 2.0, 3.0, 4.0,5.0, and 6.0 mg protein per g cellulose. All reactions with thehigh-temperature enzyme compositions were performed at 60° C., while allreactions with the Trichoderma reesei-based cellulase compositionXCL-533 were performed at 50° C.

The assay was performed as described in Example 34. The 1 ml reactionswith milled washed or milled unwashed PCS were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.Washed PCS was used at 5% total solids, whereas unwashed PCS was used at5% insoluble solids. All reactions were performed in triplicate andinvolved single mixing at the beginning of hydrolysis.

The results are shown in Table 2 and FIGS. 13A and 13B. Thehigh-temperature enzyme compositions containing Aspergillus fumigatusGH10 xyn 3 xylanase or Trichophaea saccata GH10 xylanase showed similarperformance, requiring similar protein loadings to achieve the samelevels of cellulose conversion to glucose. The improved high-temperatureenzyme compositions containing Aspergillus fumigatus GH10 xyn 3 xylanaseor Trichophaea saccata GH10 xylanase outperformed the Trichodermareesei-based cellulase composition XCL-533 on both washed (A) andunwashed (B) PCS.

TABLE 2 Protein loadings required for reaching 80% cellulose conversionof washed and unwashed PCS (mg protein per g cellulose). Temperature:60° C. for high-temperature enzyme compositions, 50° C. for XCL-533.Washed Unwashed Composition PCS PCS XCL-533 3.6 4.9 High-temperaturecomposition with Aspergillus fumigatus GH10 xyn3 xylanase 2.6 4.2High-temperature composition with Trichophaea saccata GH10 xylanase 2.54.2

Example 48: Effect of Trichoderma reesei GH3 and Talaromyces emersoniiGH3 Beta-Xylosidases on Saccharification of Milled Unwashed PCS by aHigh-Temperature Enzyme Composition at 60° C.

The ability of two beta-xylosidases, Trichoderma reesei GH3beta-xylosidase and Talaromyces emersonii GH3 beta-xylosidase, toenhance hydrolysis of milled unwashed PCS by a high-temperature enzymecomposition was evaluated at 60° C. as described in Example 34. The 1 mlreactions with milled unwashed PCS (5% insoluble solids) were conductedfor 72 hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mMmanganese sulfate. All reactions were performed in triplicate andinvolved single mixing at the beginning of hydrolysis. The results areshown in FIGS. 14A and 14B.

The high-temperature enzyme composition included 45% Aspergillusfumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 5%Trichoderma reesei Cel7B EGI, 5% Myceliophthora thermophila Cel5A EGII,5% Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity, 5% Thielavia terrestris GH61E polypeptide havingcellulolytic enhancing activity, 5% Aspergillus fumigatus GH10 xyn3xylanase, and 5% Penicillium brasilianum Cel3A beta glucosidase. Thehigh-temperature enzyme composition (3 mg total protein per g cellulose)was supplemented with Trichoderma reesei GH3 beta-xylosidase andTalaromyces emersonii GH3 beta-xylosidase at 1%, 2%, 3.5%, 5%, and 10%replacement levels, and the hydrolysis results were compared with theresults for the high-temperature enzyme composition containing nobeta-xylosidase (0% replacement level).

As shown in FIG. 14A, the inclusion of Trichoderma reesei GH3beta-xylosidase and Talaromyces emersonii GH3 beta-xylosidase in thehigh-temperature enzyme composition increased the level ofenzymatically-produced monomeric xylose from approximately 0.5 g/L (0%beta-xylosidase replacement) to approximately 2 g/L. The optimalreplacement levels were 3.5% for Trichoderma reesei GH3 beta-xylosidaseand 1-2% for Talaromyces emersonii GH3 beta-xylosidase. As shown in FIG.14B, the combined yield of glucose and cellobiose was increased by anadditional 1.5% for a mixture containing 3.5% Trichoderma reesei GH3beta-xylosidase and by an additional 2.5% for a mixture containing 2%Talaromyces emersonii GH3 beta-xylosidase.

These results demonstrated that both beta-xylosidases provided a smallbut significant benefit by increasing the degree of cellulose andhemicellulose conversion of milled unwashed PCS to soluble sugars(glucose, cellobiose and xylose). Talaromyces emersonii GH3beta-xylosidase appeared to be slightly more active than Trichodermareesei GH3 beta-xylosidase at 60° C., increasing the monomeric xyloselevel by approximately 1.5 g/L (FIG. 14A) and the cellulose conversionby an additional 2.5% (FIG. 14B) at a very low replacement level (1-2%)of the high-temperature enzyme composition.

Example 49: Evaluation of Four Cellobiohydrolases I Replacing a CBHIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of four cellobiohydrolase I proteins to replace a CBHIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 35% CBHI, 25% Myceliophthora thermophila Cel6ACBHII, 15% Thermoascus aurantiacus Cel5A EGII, 15% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Aspergillus fumigatus Cel3A beta-glucosidase, and 5% Aspergillusfumigatus GH10 xyn3 xylanase.

The following CBHI cellulases were tested in the high-temperature enzymecomposition: Trichoderma reesei Cel7A CBHI, Chaetomium thermophilumCel7A CBHI, Aspergillus fumigatus Cel7A CBHI, and Thermoascusaurantiacus Cel7A CBHI.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 15, Aspergillus fumigatus Cel7A performed better thanother cellobiohydrolases I at all temperatures from 50° C. to 65° C.Chaetomium thermophilum Cel7A also performed well in the entire range oftemperatures, but the degree of cellulose conversion to glucose waslower compared to Aspergillus fumigatus Cel7A. Trichoderma reesei Cel7Adid not perform well at temperatures above 55° C. Thermoascusaurantiacus Cel7A, which contained no CBM, showed a remarkably goodperformance at 60° C. and 65° C., but was less efficient in hydrolyzingthe cellulose in unwashed PCS at lower temperatures (50° C. and 55° C.).

Example 50: Evaluation of Four Cellobiohydrolases II Replacing a CBHIIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of several cellobiohydrolase II proteins to replace a CBHIIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 35% Aspergillus fumigatus Cel7A CBHI, 25% CBHII,15% Thermoascus aurantiacus Cel5A EGII, 15% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus Cel3A beta-glucosidase, and 5% Aspergillus fumigatus GH10 xyn3xylanase.

The following CBHII cellulases were tested in the high-temperatureenzyme composition: Myceliophthora thermophila Cel6A CBHII, Thielaviaterrestris Cel6A CBHII, Aspergillus fumigatus Cel6A CBHII, andTrichophaea saccata Cel6A CBHII.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 16, CBHII from Aspergillus fumigatus performed aboutthe same as CBHII from Myceliophthora thermophila at all temperatures,showing only a slightly lower hydrolysis at 60-65° C. Thielaviaterrestris Cel6A CBHII had high thermostability and performed well inthe entire range of temperatures, but the degree of cellulose conversionto glucose was lower compared to Myceliophthora thermophila Cel6A CBHII.Trichophaea saccata Cel6A CBHII had high activity at 50-55° C., but didnot perform well at higher temperatures.

Example 51: Evaluation of Two Endoglucanases I Replacing anEndoglucanase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

The ability of two endoglucanase I proteins to replace an endoglucanasecomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 35% Aspergillus fumigatus Cel7A CBHI, 25%Myceliophthora thermophila Cel6A CBHII, 15% EG cellulase, 15%Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity, 5% Aspergillus fumigatus Cel3A beta-glucosidase, and 5%Aspergillus fumigatus GH10 xyn3 xylanase.

The following EGIs were tested in the high-temperature enzymecomposition: Trichoderma reesei Cel7B EGI and Aspergillus terreus Cel7EGI.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 17, the high-temperature enzyme composition containingAspergillus terreus Cel7 EGI performed significantly better than thehigh-temperature enzyme composition containing Trichoderma reesei Cel7BEGI within this temperature range.

Example 52: Evaluation of Three Endoglucanases II Replacing anEndoglucanase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

The ability of three endoglucanase II proteins to replace anendoglucanase component in a high-temperature enzyme composition (3 mgtotal protein per g cellulose) was tested at 50° C., 55° C., 60° C., and65° C. using milled unwashed PCS as a substrate. The high-temperatureenzyme composition included 35% Aspergillus fumigatus Cel7A CBHI, 25%Myceliophthora thermophila Cel6A CBHII, 15% EG cellulase, 15%Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity, 5% Aspergillus fumigatus Cel3A beta-glucosidase, and 5%Aspergillus fumigatus GH10 xyn3 xylanase.

The following EGIIs were tested in the high-temperature enzymecomposition: Trichoderma reesei Cel5A EGII, Myceliophthora thermophilaCel5A EGII, and Thermoascus aurantiacus Cel5A EGII.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 18, all endoglucanase II proteins performed similarlywithin this temperature range, with Myceliophthora thermophila Cel5AEGII slightly outperforming Trichoderma reesei Cel5A EGII andThermoascus aurantiacus Cel5A EGII.

Example 53: Evaluation of Three Beta-Glucosidases Replacing aBeta-Glucosidase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

In a first experiment, three beta-glucosidases, including Aspergillusfumigatus Cel3A beta-glucosidase, Penicillium brasilianum Cel3Abeta-glucosidase, and Aspergillus niger Cel3 beta-glucosidase, wereevaluated in a high-temperature enzyme composition at 50° C., 55° C.,and 60° C. using milled unwashed PCS as a substrate. Thehigh-temperature enzyme composition included 45% Aspergillus fumigatusCel7A CBHI, 25% Thielavia terrestris Cel6A CBHII, 5% Trichoderma reeseiCel7B EGI, 5% Thermoascus aurantiacus Cel5A EGII, 5% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Thielavia terrestris GH61E polypeptide having cellulolytic enhancingactivity, 5% Aspergillus fumigatus GH10 xyn3 xylanase, and 5%beta-glucosidase. The high-temperature enzyme composition was used at3.0 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 19 demonstrated that all threebeta-glucosidases performed about the same at 50° C. and 55° C., whereasAspergillus niger Cel3 beta-glucosidase showed slightly lower hydrolysisat 60° C. than the other two beta-glucosidases.

In a second experiment, Aspergillus fumigatus Cel3A beta-glucosidase andPenicillium brasilianum Cel3A beta-glucosidase were compared in ahigh-temperature enzyme composition at four temperatures, 50° C., 55°C., 60° C., and 65° C., using milled unwashed PCS as a substrate. Thehigh-temperature enzyme composition included 35% Aspergillus fumigatusCel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 15% Thermoascusaurantiacus Cel5A EGII, 15% Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity, 5% beta-glucosidase, and 5%Aspergillus fumigatus GH10 xyn3 xylanase. The high-temperature enzymecomposition was used at 3.0 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 20 demonstrated that Cel3A beta-glucosidasesfrom Aspergillus fumigatus and Penicillium brasilianum performed aboutthe same within this temperature range.

Example 54: Evaluation of Six Xylanases Replacing a Xylanase Componentin a High-Temperature Enzyme Composition in Saccharification of MilledUnwashed PCS at 50-65° C.

The ability of six xylanases to replace a xylanase component in ahigh-temperature enzyme composition (3 mg total protein per g cellulose)was tested at 50° C., 55° C., 60° C., and 65° C. using milled unwashedPCS as a substrate. The high-temperature enzyme composition included 35%Aspergillus fumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6ACBHII, 15% Thermoascus aurantiacus Cel5A EGII, 15% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Aspergillus fumigatus Cel3A beta-glucosidase, and 5% xylanase.

The following xylanases were tested in the high-temperature enzymecomposition: Aspergillus aculeatus GH10 xyn II xylanase, Aspergillusfumigatus GH10 xyn3, Trichophaea saccata GH10 xylanase, Thermobifidafusca GH11 xylanase, Penicillium pinophilum GH10 xylanase, and Thielaviaterrestris GH10E xylanase.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 21, GH10 xylanase from Penicillium pinophilum wassuperior to GH10 xylanases from Aspergillus fumigatus and Trichophaeasaccata at all temperatures from 50° C. to 65° C. The Aspergillusaculeatus GH10 and Thielavia terrestris GH10E xylanases performed aboutthe same as the Aspergillus fumigatus GH10 and Trichophaea saccata GH10xylanases at 50° C. and 55° C., but did not perform well at highertemperatures (60° C. and 65° C.). The Thermobifida fusca GH11 performedrelatively well at 60° C. and 65° C., but overall the degree ofcellulose conversion to glucose was lower compared to other xylanases.

Example 55: Evaluation of the Ability of Four GH61 Polypeptides HavingCellulolytic Enhancing Activity to Enhance PCS-Hydrolyzing Activity of aHigh-Temperature Enzyme Composition at 50-65° C. Using Milled UnwashedPCS

The ability of four GH61 polypeptides having cellulolytic enhancingactivity, Thermoascus aurantiacus GH61A, Thielavia terrestris GH61E,Penicillium pinophilum GH61, and Aspergillus fumigatus GH61B, to enhancethe PCS-hydrolyzing activity of a high-temperature enzyme compositionwas evaluated using milled unwashed PCS at 50° C., 55° C., 60° C., and65° C. The high-temperature enzyme composition included 35% Aspergillusfumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6A CBHII, 15%Thermoascus aurantiacus Cel5A EGII, 15% GH61 polypeptide havingcellulolytic enhancing activity, 5% Aspergillus fumigatus Cel3Abeta-glucosidase, and 5% Aspergillus fumigatus GH10 xyn3 xylanase. Thehigh-temperature enzyme composition was used at 3 mg total protein per gcellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 22, all four GH61 polypeptides showed a significantcellulase-enhancing activity, with Thermoascus aurantiacus GH61Apolypeptide being the most efficient enhancer among the four within thistemperature range. High-temperature enzyme compositions containingThermoascus aurantiacus GH61A and Thielavia terrestris GH61Epolypeptides performed better at higher temperatures (60° C. and 65° C.)than high-temperature enzyme compositions containing Penicilliumpinophilum GH61 and Aspergillus fumigatus GH61B polypeptides.

Example 56: Evaluation of the Ability of Three GH61 Polypeptides HavingCellulolytic Enhancing Activity to Enhance PCS-Hydrolyzing Activity of aHigh-Temperature Enzyme Composition at 50-65° C. Using Milled UnwashedPCS

The ability of three GH61 polypeptides having cellulolytic enhancingactivity, Thermoascus aurantiacus GH61A, Thielavia terrestris GH61N, andPenicillium sp GH61A, to enhance the PCS-hydrolyzing activity of ahigh-temperature enzyme composition was evaluated using milled unwashedPCS at 50° C., 55° C., 60° C., and 65° C. The high-temperature enzymecomposition included 45% Aspergillus fumigatus Cel7A CBHI, 25%Myceliophthora thermophila Cel6A CBHII, 10% Myceliophthora thermophilaCel5A EGII, 10% GH61 polypeptide, 5% Aspergillus fumigatus Cel3Abeta-glucosidase, and 5% Aspergillus fumigatus GH10 xyn3 xylanase. Theresults for the enzyme compositions containing GH61 polypeptides (3 mgtotal protein per g cellulose) were compared with the results for asimilar enzyme composition to which no GH61 polypeptide was added (2.7mg total protein per g cellulose).

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 23, all three GH61 polypeptides showed a significantcellulase-enhancing activity, with Thermoascus aurantiacus GH61Apolypeptide being the most efficient enhancer at 55-65° C., andThielavia terrestris GH61N polypeptide slightly outperformingThermoascus aurantiacus GH61A polypeptide at 50° C. The high-temperatureenzyme composition containing Penicillium sp. GH61A polypeptideperformed almost as well as the high-temperature enzyme compositioncontaining Thermoascus aurantiacus GH61A polypeptide at all fourtemperatures (50-65° C.). Thielavia terrestris GH61N polypeptideperformed well at 50° C. and 55° C., but showed a significant decline inperformance at higher temperatures (60° C. and 65° C.).

Example 57: Hydrolysis of Milled Unwashed PCS by Trichodermareesei-Based XCL-602 Cellulase at Different Replacement Levels byAspergillus fumigatus Cel7A Cellobiohydrolase I, Myceliophthorathermophila Cel6A Cellobiohydrolase II, or their Binary Compositions at50-60° C.

A Trichoderma reesei strain 981-08-D4-based cellulase containingAspergillus fumigatus beta-glucosidase and Thermoascus aurantiacus GH61Apolypeptide, designated Trichoderma reesei-based XCL-602 cellulase, wastested in 1-ml hydrolysis reactions at 50° C., 55° C., and 60° C. usingmilled unwashed PCS as a substrate. The Trichoderma reesei-based XCL-602cellulase was used alone or in mixtures with Aspergillus fumigatus Cel7ACBHI (10%, 20%, 30% or 40% of total protein), Myceliophthora thermophilaCel6A CBHII (10% or 20% of total protein), or both Aspergillus fumigatusCel7A CBHI and Myceliophthora thermophila Cel6A CBHII (10%/10%, 10%/20%,20%/10%, 20%/20%, 30%/10%, 30%/20%, 40%/20% of total protein). The levelof Thermoascus aurantiacus GH61A polypeptide in all Trichoderma reeseiXCL-602 compositions was maintained constant at 8% of total protein.Trichoderma reesei-based enzyme composition SaMe-MF268 (XCL-533) wasalso included in the experiment. The non-replaced Trichodermareesei-based XCL-602 cellulase and Trichoderma reesei-based XCL-533cellulase and various Trichoderma reesei XCL-602 compositions containingAspergillus fumigatus Cel7A CBHI and/or Myceliophthora thermophila Cel6ACBHII were used at 3 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

The results shown in FIG. 24 demonstrated that non-replaced Trichodermareesei-based XCL-602 cellulase performed about the same as non-replacedTrichoderma reesei-based XCL-533 cellulase at all three temperatures. At50° C. and 55° C., the replacement of 10%, 20%, or 30% protein inXCL-602 cellulase by Aspergillus fumigatus Cel7A CBHI or the replacementof 10% or 20% protein in XCL-602 cellulase by Myceliophthora thermophilaCel6A CBHII did not have a significant effect on the degree of celluloseconversion to glucose after 72 hours of hydrolysis. The replacement of40% protein in the Trichoderma reesei-based XCL-602 cellulase byAspergillus fumigatus Cel7A CBHI had a negative effect on the degree ofcellulose conversion to glucose at 50° C. and 55° C. At 60° C., thereplacement of protein in the Trichoderma reesei-based XCL-602 cellulaseby Aspergillus fumigatus Cel7A CBHI (10-40% of total protein) orMyceliophthora thermophila Cel6A CBHII (10-20% of total protein)significantly improved the hydrolysis over non-replaced Trichodermareesei-based XCL-602 cellulase at an equivalent protein loading (3 mgprotein per g cellulose). Higher replacement levels by Aspergillusfumigatus Cel7A CBHI or Myceliophthora thermophila Cel6A CBHII provideda greater hydrolysis enhancement over non-replaced XCL-602 cellulase at60° C.

For the Trichoderma reesei-based XCL-602 cellulase compositions thatincluded both Aspergillus fumigatus Cel7A CBHI and Myceliophthorathermophila Cel6A CBHII, the optimal replacement levels were 10-20%Aspergillus fumigatus Cel7A CBHI/10-20% Myceliophthora thermophila Cel6ACBHII at 50° C. and 55° C., and 40% Aspergillus fumigatus Cel7A CBHI/20%Myceliophthora thermophila Cel6A CBHII at 60° C.

Example 58: Hydrolysis of Milled Unwashed PCS by Trichodermareesei-Based XCL-602 Cellulase Containing Aspergillus fumigatus Cel7ACellobiohydrolase I and Myceliophthora Thermophila Cel6ACellobiohydrolase II, and Additionally Supplemented by AspergillusFumigatus GH10 Xyn 3 and/or Thielavia terrestris GH61E at 50-60° C.

The Trichoderma reesei-based XCL-602 cellulase containing Aspergillusfumigatus Cel7A cellobiohydrolase I and Myceliophthora thermophila Cel6Acellobiohydrolase II was tested in 1-ml hydrolysis reactions at 50° C.,55° C. and 60° C. using milled unwashed PCS as a substrate. TheTrichoderma reesei-based XCL-602 cellulase was used alone (3.0 mgprotein per g cellulose) or with replacement by different binarycompositions consisting of Aspergillus fumigatus Cel7A CBHI andMyceliophthora thermophila Cel6A CBHII (10%/10% or 40%/20% of totalprotein) to a total protein loading of 3 mg protein per g cellulose. Thelevel of Thermoascus aurantiacus GH61A polypeptide in all Trichodermareesei XCL-602-based compositions containing Aspergillus fumigatus Cel7ACBHI and Myceliophthora thermophila Cel6A CBHII was maintained constantat 8% of total protein. The Trichoderma reesei XCL-602-basedcompositions containing Aspergillus fumigatus Cel7A CBHI andMyceliophthora thermophila Cel6A CBHII (3 mg protein per g cellulose)were additionally supplemented with 5% Aspergillus fumigatus GH10 xyn 3xylanase (0.15 mg protein per g cellulose), 5% Thielavia terrestrisGH61E polypeptide (0.15 mg protein per g cellulose), or with a binarycomposition consisting of 5% Aspergillus fumigatus GH10 xyn 3 xylanaseand 5% Thielavia terrestris GH61E polypeptide (0.30 mg protein per gcellulose). For comparison, the non-replaced Trichoderma reesei-basedXCL-602 cellulase was used alone at 3.15 mg protein per g cellulose and3.3 mg protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

The results shown in FIGS. 25A and 25B demonstrated that the addition of5% Aspergillus fumigatus GH10 xyn3 xylanase and/or 5% Thielaviaterrestris GH61E polypeptide to the Trichoderma reesei XCL-602-basedcompositions containing Aspergillus fumigatus Cel7A CBHI andMyceliophthora thermophila Cel6A CBHII significantly improved thehydrolysis performance at all three temperatures, with a very largeimprovement obtained when both the Aspergillus fumigatus GH10 xyn3xylanase and the Thielavia terrestris GH61E polypeptide were addedtogether. The best XCL-602-based composition in this experiment (XCL-602with 10% replacement by Aspergillus fumigatus Cel7A CBHI and 10%replacement by Myceliophthora thermophila Cel6A CBHII, and withadditional supplementation by 5% Aspergillus fumigatus GH10 xyn3xylanase and 5% Thielavia terrestris GH61E polypeptide) required 3.3 mgprotein per g cellulose to achieve 82% conversion of cellulose toglucose in milled unwashed PCS after 72 hours of hydrolysis at 55° C.This represents a 1.5× reduction in protein loading compared toTrichoderma reesei XCL-533 (SaMe-MF268), which required 4.9 mg proteinper g cellulose to achieve 80% conversion of cellulose to glucose inmilled unwashed PCS after 72 hours of hydrolysis at 50° C. (Table 2).

Example 59: Hydrolysis of Milled Unwashed PCS by Trichodermareesei-Based XCL-602 Cellulase Compositions Containing DifferentReplacement Levels of Trichoderma reesei-Based XCL-592 Cellulase at50-60° C.

The Trichoderma reesei-based XCL-602 cellulase was tested alone (3.0 mgprotein per g cellulose) or in mixtures with Trichoderma reeseiRutC30-based cellulase containing Aspergillus aculeatus GH10 xylanase,designated as Trichoderma reesei-based XCL-592 cellulase. TheTrichoderma reesei-based XCL-592 cellulase replaced 5%, 10%, 15%, 20%,or 25% of protein in the Trichoderma reesei-based XCL-602 cellulase to atotal protein loading of 3 mg protein per g cellulose. The non-replacedTrichoderma reesei-based XCL-602 cellulase and XCL-602-based enzymecompositions were tested in 1-ml hydrolysis reactions at 50° C., 55° C.and 60° C. using milled unwashed PCS as a substrate. For comparison,Trichoderma reesei-based XCL-533 cellulase was tested at 4.5 mg proteinper g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

The results shown in FIG. 26 demonstrated that the optimal replacementlevel of Trichoderma reesei-based XCL-602 cellulase by Trichodermareesei-based XCL-592 cellulase at 50° C. and 55° C. was between 10% and15% of total protein. At 50° C. and 55° C., the best Trichodermareesei-based XCL-602 cellulase compositions performed significantlybetter than Trichoderma reesei-based XCL-602 cellulase alone at anequivalent protein loading (3 mg protein per g cellulose), but were notable to reach the performance level obtained with Trichodermareesei-based XCL-533 cellulase at 4.5 mg protein per g cellulose.

Example 60: Comparison of Thermoascus aurantiacus GH61A and Thielaviaterrestris GH61E GH61 Polypeptides Having Cellulolytic EnhancingActivity Replacing 5% of Protein in Trichoderma reesei-Based XCL-602Cellulase or XCL-602-Based Enzyme Composition in Hydrolysis of MilledUnwashed PCS at 50-60° C.

The Trichoderma reesei-based XCL-602 cellulase was tested alone and with5% replacement by Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity or Thielavia terrestris GH61Epolypeptide having cellulolytic enhancing activity at 50-60° C. usingmilled unwashed PCS as a substrate. In addition, the Trichodermareesei-based XCL-602 cellulase composition containing 30% Aspergillusfumigatus Cel7A CBHI, 20% Myceliophthora thermophila Cel6A CBHI,I and 5%Thermoascus aurantiacus GH61A polypeptide was tested in comparison witha similar composition containing Thielavia terrestris GH61E polypeptideinstead of Thermoascus aurantiacus GH61A polypeptide. All compositionswere tested at 3 mg protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

As shown in FIG. 27A, replacement of 5% protein in Trichodermareesei-based XCL-602 cellulase by Thielavia terrestris GH61E polypeptidedid not provide an advantage over the equivalent replacement byThermoascus aurantiacus GH61A polypeptide. Similarly, as shown in FIG.27B, the replacement of 5% protein in Trichoderma reesei-based XCL-602cellulase by Thielavia terrestris GH61E polypeptide along with the 30%replacement by Aspergillus fumigatus Cel7A CBHI and 20% replacement byMyceliophthora thermophila Cel6A CBHII, did not provide an advantageover the equivalent replacement by Thermoascus aurantiacus GH61Apolypeptide. Each GH61 polypeptide was tested at six differentreplacement levels ranging from 5% to 20%, and the conclusion wasconsistent for all replacement levels (data not shown). The resultsindicated that the Thielavia terrestris GH61E polypeptide did notsynergize with the Thermoascus aurantiacus GH61A polypeptide under theseconditions.

Example 61: Comparison of XCL-602-Based Enzyme Compositions ContainingDifferent Replacement Levels of Thermoascus aurantiacus GH61A orThielavia terrestris GH61E GH61 Polypeptides Having CellulolyticEnhancing Activity in Hydrolysis of Milled Unwashed PCS at 50-60° C.

Thermoascus aurantiacus GH61A and Thielavia terrestris GH61E GH61polypeptides having cellulolytic enhancing activity were testedseparately at six different replacement levels (5%, 7.5%, 10%, 12.5%,15%, and 20%) in different Trichoderma reesei-based XCL-602 cellulasecompositions at 50-60° C. using milled unwashed PCS as a substrate. Inone case, the Trichoderma reesei-based XCL-602 cellulase compositionscomprised different replacement levels of a GH61 polypeptide along witha 5% replacement by Aspergillus fumigatus GH10 xyn3 xylanase. In anothercase, Trichoderma reesei-based XCL-602 cellulase compositions compriseddifferent replacement levels of a GH61 polypeptide along with a 30%replacement by Aspergillus fumigatus Cel7A CBHI, a 20% replacement byMyceliophthora thermophila Cel6A CBHII, and a 5% replacement byAspergillus fumigatus GH10 xyn3 xylanase. All enzyme compositions weretested at 3 mg total protein per g cellulose. For comparison,Trichoderma reesei-based XCL-533 cellulase was tested at 4.5 mg proteinper g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

The results shown in Table 3 demonstrated that the optimal replacementlevel for both GH61 polypeptides at 50° C. and 55° C. was 10-12.5% ofthe total protein in the Trichoderma reesei-based XCL-602 cellulasecompositions. At 60° C., the optimal replacement level for both GH61polypeptides was 20% of the total protein in the Trichodermareesei-based XCL-602 cellulase compositions. The equivalent replacementlevels of the Thermoascus aurantiacus GH61A and Thielavia terrestrisGH61E polypeptides in similar Trichoderma reesei-based XCL-602 cellulasecompositions provided a similar enhancement of the cellulose hydrolysisin milled unwashed PCS at 50-60° C.

TABLE 3 Comparison of Trichoderma reesei-based XCL-602 cellulasecompositions containing different replacement levels of Thermoascusaurantiacus GH61A or Thielavia terrestris GH61E polypeptidesinhydrolysis of milled unwashed PCS at 50-60° C. GH61 replacement level50° C. 55° C. 60° C. XCL-602 with 5% replacement by Aspergillusfumigatus GH10 xyn3 xylanase and 0-20% replacement by a GH61 polypeptide(3 mg total protein per g cellulose)  0% T. aurantiacus GH61A 67% 66%42%  5% T. aurantiacus GH61A 69% 69% 46%  7.5% T. aurantiacus GH61A 69%69% 47% 10% T. aurantiacus GH61A 69% 69% 49% 12.5% T. aurantiacus GH61A69% 69% 49% 15% T. aurantiacus GH61A 69% 69% 49% 20% T. aurantiacusGH61A 66% 69% 51%  0% T. terrestris GH61E 67% 66% 42%  5% T. terrestrisGH61E 68% 69% 45%  7.5% T. terrestris GH61E 68% 68% 46% 10% T.terrestris GH61E 68% 68% 47% 12.5% T. terrestris GH61E 65% 68% 48% 15%T. terrestris GH61E 64% 67% 48% 20% T. terrestris GH61E 62% 65% 49%XCL-602 with 30% replacement by Aspergillus fumigatus Cel7A CBHI, 20%replacement by Myceliophthora thermophila Cel6A CBHII, 5% replacement byAspergillus fumigatus GH10 xyn3 xylanase and 0-20% replacement by a GH61polypeptide (3 mg total protein per g cellulose)  0% T. aurantiacusGH61A 64% 68% 58%  5% T. aurantiacus GH61A 70% 75% 66%  7.5% T.aurantiacus GH61A 72% 75% 67% 10% T. aurantiacus GH61A 72% 76% 68% 12.5%T. aurantiacus GH61A 71% 76% 70% 15% T. aurantiacus GH61A 71% 76% 69%20% T. aurantiacus GH61A 68% 72% 71%  0% T. terrestris GH61E 64% 68% 58% 5% T. terrestris GH61E 68% 72% 65%  7.5% T. terrestris GH61E 69% 73%65% 10% T. terrestris GH61E 69% 74% 69% 12.5% T. terrestris GH61E 67%73% 68% 15% T. terrestris GH61E 66% 74% 69% 20% T. terrestris GH61E 65%72% 69%

Example 62: Hydrolysis of Milled Unwashed PCS by Non-Replaced XCL-602and Various XCL-602-Based Enzyme Compositions (3 mg Protein Per gCellulose) in Comparison with Trichoderma reesei-Based XCL-533 Cellulase(4.5 mg Protein Per g Cellulose) at 50-60° C.

Example 61 was repeated using the same Trichoderma reesei-based XCL-602cellulase compositions except that only one GH61 polypeptide(Thermoascus aurantiacus GH61A) was tested at a single replacement level(10% of total protein). The Trichoderma reesei-based XCL-602 cellulaseand all Trichoderma reesei-based XCL-602 cellulase compositions wereused at 3 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith 50 mg of insoluble PCS solids per ml were conducted for 72 hours in50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

The results shown in FIG. 28A demonstrated that the replacement ofTrichoderma reesei-based XCL-602 cellulase with a composition comprising5% Aspergillus fumigatus GH10 xyn3 xylanase and 10% Thermoascusaurantiacus GH61A polypeptide to a total protein loading of 3 mg proteinper g cellulose has significantly improved the hydrolysis yield over thenon-replaced Trichoderma reesei-based XCL-602 cellulase at all threetemperatures. As shown in FIG. 27B, the enhancement was even morepronounced when the Trichoderma reesei-based XCL-602 cellulase wasadditionally replaced with 30% Aspergillus fumigatus Cel7A CBHI and 20%Myceliophthora thermophila Cel6A CBHII. The results shown in FIG. 28Bdemonstrated that the replacement of Trichoderma reesei-based XCL-602cellulase with 30% Aspergillus fumigatus Cel7A CBHI and 20%Myceliophthora thermophila Cel6A CBHII increased the degree of celluloseconversion to glucose after 72 hours of hydrolysis at 60° C. from 37% to48%. Additional 5% replacement by Aspergillus fumigatus GH10 xyn3xylanase increased the degree of cellulose conversion to glucose to 58%,and additional 10% replacement by Thermoascus aurantiacus GH61Apolypeptide increased the degree of cellulose conversion to glucose to68% compared to 37% obtained with non-replaced Trichoderma reesei-basedXCL-602 cellulase. At 3 mg protein per g cellulose, the bestXCL-602-based enzyme composition comprising the XCL-602 cellulase with30% replacement by Aspergillus fumigatus Cel7A CBHI, 20% replacement byMyceliophthora thermophila Cel6A CBHII, 5% replacement by Aspergillusfumigatus GH10 xyn3 xylanase and 10% replacement by Thermoascusaurantiacus GH61A polypeptide, was capable of achieving the samecellulose conversion of milled unwashed PCS at 55° C. (76%) asnon-replaced Trichoderma reesei-based XCL-602 cellulase at 4.5 mgprotein per g cellulose and 50° C.-a 1.5-fold reduction in proteinloading.

Example 63: Preparation of Penicillium emersonii Strain NN051602 GH7Cellobiohydrolase I

The Penicillium emersonii strain NN051602 Cel7 cellobiohydrolase I (SEQID NO: 157 [DNA sequence] and SEQ ID NO: 158 [deduced amino acidsequence]) was obtained according to the procedure described below.

Penicillium emersonii was grown on a PDA agar plate at 45° C. for 3days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Oligonucleotide primers, shown below, were designed to amplify the GH7cellobiohydrolase I gene from genomic DNA of Penicillium emersonii. AnIN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 207)5′-ACACAACTGGGGATCCACC atgcttcgacgggctcttc-3′ Antisense primer:(SEQ ID NO: 208) 5′-GTCACCCTCTAGATCT CGCAGAGCAACTTCCGTCTACTTC-3′Bold letters represented the coding sequence (for the sense primer) orthe downstream sequence of the coding region (for the antisense primer).The remaining sequence was homologous to the insertion sites ofpPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium emersonii genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and0.6 unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of50 μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minute; 8 cycles of denaturingat 98° C. for 15 seconds, annealing at 65° C. for 30 seconds, with 1° C.decrease per cycle and elongation at 72° C. for 80 seconds; and another23 cycles each at 98° C. for 15 seconds, 66° C. for 30 seconds and 72Cfor 75 seconds; final extension at 72° C. for 7 minutes. The heat blockthen went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where anapproximately 1.4 kb product band was excised from the gel, and purifiedusing an ILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit accordingto the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in pGH7_ZY209383 in whichtranscription of the Penicillium emersonii GH7 cellobiohydrolase I genewas under the control of the Aspergillus oryzae TAKA amylase promoterfrom the gene for Aspergillus oryzae alpha-amylase. The cloningoperation was according to the manufacturer's instruction. In brief, 30ng of pPFJO355 digested with Barn I and Bgl II, and 60 ng of thePenicillium emersonii GH7 cellobiohydrolase I gene purified PCR productwere added to the reaction vial and resuspended the powder in a finalvolume of 10 ul with addition of deionized water. The reaction wasincubated at 37° C. for 15 minutes and then 50° C. for 15 minutes. Fiveμl of the reaction were used to transform E. coli TOP10 competent cells.An E. coli transformant containing pGH7_ZY209383 was detected by colonyPCR and plasmid DNA was prepared using a QIAprep Spin Miniprep Kit(QIAGEN Inc., Valencia, Calif., USA). The Penicillium emersonii GH7cellobiohydrolase I gene insert in pGH7_ZY209383 was confirmed by DNAsequencing using a 3730XL DNA Analyzer.

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pGH7_ZY209383. The transformationyielded about 50 transformants. Twelve transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT® Blue. SDS-PAGE profiles of the cultures showedthat the majority of the transformants had a major smeary band ofapproximately 50 kDa. The expression strain was designated as A. oryzaeEXP03477.

A slant of A. oryzae EXP03477 was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

A 1600 ml volume of filtered broth of A. oryzae EXP03477 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml of 25 mM Bis-Tris pH 6.5 buffer, dialyzed against the same buffer,and filtered through a 0.45 μm filter; the final volume was 200 ml. Thesolution was applied to a 40 ml Q Sepharose® Fast Flow columnequilibrated with 25 mM Bis-Tris pH 6.5, and the proteins were elutedwith a linear NaCl gradient (0-0.4 M). Fractions with activity againstPASC were collected and applied to a 40 ml Phenyl Sepharose™ HIC column(GE Healthcare, Piscataway, N.J., USA) equilibrated in 20 mM PBS with1.8 M (NH₄)₂SO₄ pH 7 buffer, and the proteins were eluted with 20 mM PBSpH 7. Fractions from the column with activity toward phosphoric acidswollen cellulose (PASC) as substrate were analyzed by SDS-PAGE using aNUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer. Fractions with thecorrect molecular weight were pooled. Then the pooled solution wasconcentrated by ultrafiltration. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 64: Preparation of Penicillium Pinophilium Strain NN046877 GH7Cellobiohydrolase I

The Penicillium pinophilium strain NN046877 Cel7 cellobiohydrolase II(SEQ ID NO: 159 [DNA sequence] and SEQ ID NO: 160 [deduced amino acidsequence]) was obtained according to the procedure described below.

Penicillium pinophilum was grown on a PDA agar plate at 37° C. for 4-5days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Mini Kit.

Oligonucleotide primers, shown below, were designed to amplify the GH7cellobiohydrolase I gene from the genomic DNA of Penicillium pinophilum.An IN-FUSION™ CF Dry-down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 209)5′-ACACAACTGGGGATCCACCATGTCTGCCTTGAACTCTTTC-3′ Antisense primer:(SEQ ID NO: 210) 5′-GTCACCCTCTAGATCTTCACAAACATTGAGAGTAGTAAGGGTT-3′Bold letters represented the coding sequence and the remaining sequencewas homologous to the insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium pinophilum genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and0.6 unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of50 μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minute; 5 cycles of denaturingat 98° C. for 15 seconds, annealing at 56° C. for 30 seconds, with 1° C.increasing per cycle and elongation at 72° C. for 75 seconds; andanother 25 cycles each at 98° C. for 15 seconds, 65C for 30 seconds and72° C. for 75 seconds; final extension at 72° C. for 10 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where anapproximately 1.6 kb product band was excised from the gel, and purifiedusing an ILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit accordingto the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in pPpin6 in whichtranscription of the Penicillium pinophilum GH7 cellobiohydrolase I genewas under the control of the Aspergillus oryzae TAKA amylase promoter.In brief, 30 ng of pPFJO355 digested with Barn I and Bgl II, and 100 ngof the Penicillium pinophilum GH7 cellobiohydrolase I gene purified PCRproduct were added to a reaction vial and resuspended in a final volumeof 10 μl with addition of deionized water. The reaction was incubated at37° C. for 15 minutes and then 50° C. for 15 minutes. Three μl of thereaction were used to transform E. coli TOP10 competent cells. An E.coli transformant containing pPpin6 was detected by colony PCR andplasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc.,Valencia, Calif., USA). The Penicillium pinophilum GH7 cellobiohydrolaseI gene insert in pPpin6 was confirmed by DNA sequencing using a 3730XLDNA Analyzer.

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pPpin6. The transformationyielded about 50 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT® Blue. SDS-PAGE profiles of the cultures showedthat the majority of the transformants had a major smear band ofapproximately 60-90 kDa. The expression strain was designated as A.oryzae EXP02768.

A slant of A. oryzae EXP02768 was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

A 1600 ml volume of the filtered broth of A. oryzae EXP02768 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml of 25 mM Bis-Tris pH 6.0, dialyzed against the same buffer, andfiltered through a 0.45 μm filter; the final volume was 200 ml. Thesolution was applied to a 40 ml Q Sepharose® Fast Flow columnequilibrated in 25 mM Bis-Tris pH 6.0, and the proteins were eluted witha linear NaCl gradient (0-0.4 M). Fractions from the column withactivity against pNP-β-D-lactopyranoside were analyzed by SDS-PAGE usinga NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer. Fractions with thecorrect molecular weight were pooled and concentrated byultrafiltration. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 65: Preparation of Aspergillus terreus ATCC 28865 GH7Cellobiohydrolase I

The Aspergillus terreus ATCC 28865 GH7 cellobiohydrolase I (SEQ ID NO:161 [DNA sequence] and SEQ ID NO: 162 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the cellobiohydrolase I gene from Aspergillus terreus ATCC 28865genomic DNA. Genomic DNA was isolated using a FastDNA spin for Soil Kit(MP Biomedicals, OH, USA).

Primer #222: (SEQ ID NO: 211) 5′-TAAGAATTCACCATGCCTTCCACCTACGA-3′Primer #302: (SEQ ID NO: 212) 5′-TATGCGGCCGCATTCTCCTAGACACCCCGCAT-3′

The amplification reaction was composed of 1 μl of Aspergillus terreusgenomic DNA, 12.5 μl of 2× REDDYMIX™ PCR Buffer (Thermo FisherScientific Inc., Waltham, Mass., USA), 1 μl of 5 μM primer #222, 1 μl of5 μM primer #302, and 9.5 μl of H2O. The amplification reaction wasincubated in a PTC-200 DNA ENGINE™ Thermal Cycler programmed for 1 cycleat 94° C. for 2 minutes; and 35 cycles each at 94° C. for 15 seconds and60° C. for 1.5 minutes.

A 1.7 kb PCR reaction product was isolated by 1% agarose gelelectrophoresis using TAE buffer and staining with SYBR Safe DNA gelstain (Invitrogen Corp., Carlsbad, Calif., USA). The DNA band wasvisualized with the aid of an Eagle Eye Imaging System and a DarkReaderTransilluminator (Clare Chemical Research, Dolores, Colo., USA). The 1.7kb DNA band was excised from the gel and purified using a GFX® PCR DNAand Gel Band Purification Kit according to the manufacturer'sinstructions.

The 1.7 kb fragment was cleaved with EcoR I and Not I and purified usinga GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The cleaved 1.7 kb fragment was then directionally cloned by ligationinto Eco RI-Not I cleaved pXYG1051 (WO 2005/080559) using T4 ligase(Promega, Madison, Wis., USA) according to the manufacturer'sinstructions. The ligation mixture was transformed into E. coli TOP10Fcompetent cells according to the manufacturer's instructions. Thetransformation mixture was plated onto LB plates supplemented with 100μg of ampicillin per ml. Plasmid minipreps were prepared from severaltransformants and sequenced. One plasmid with the correct Aspergillusterreus GH7 coding sequence was chosen. The plasmid was designated pXYG1051-N P003568.

The expression plasmid pXYG1051-NP003568 was transformed intoAspergillus oryzae JaL355 as described in WO 98/00529. Transformantswere purified on selection plates through single conidia prior tosporulating them on PDA plates. Production of the Aspergillus terreusGH7 polypeptide by the transformants was analyzed from culturesupernatants of 1 ml 96 deep well stationary cultivations at 26° C. inYP medium with 2% maltodextrin. Expression was verified by SDS-PAGEusing a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer and Coomassieblue staining. One transformant was selected for further work anddesignated Aspergillus oryzae 64.1.

For larger scale production, Aspergillus oryzae 64.1 spores were spreadonto a PDA plate and incubated for five days at 37° C. The confluentspore plate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspension was then used toinoculate twenty-five 500 ml flasks containing 100 ml of YPG medium. Theculture was incubated at 30° C. with constant shaking at 120 rpm. At dayfour post-inoculation, the culture broth was collected by filtrationthrough a triple layer of Whatman glass microfiber filters of 1.6 μm,1.2 μm, and 0.7 μm. Fresh culture broth from this transformant produceda band of GH7 protein of approximately 55 kDa. The identity of this bandas the Aspergillus terreus GH7 polypeptide was verified by peptidesequencing.

Two liters of the filtered broth were concentrated to 400 ml and washedwith 50 mM HEPES pH 7.0 using a SARTOFLOW® Alpha ultrafiltration systemwith a 10 kDa MW-CO (Sartorius Stedim Biotech S.A., Aubagne Cedex,France). Ammonium sulphate was added to a final concentration of 1 M anddissolved in the ultrafiltrate. The solution was applied on a Source 15Phenyl XK 26/20 50 ml column (GE Healthcare, HiHerod, Denmark). Afterapplication the column was washed with 150 ml of 1 M ammonium sulphateand eluded with 1 column volume of 50% ethanol in a 0% to 100% gradientfollowed by 5 column volumes of 50% ethanol at a flow rate of 10ml/minute. Fractions of 10 ml were collected and analyzed by SDS-PAGE.Fraction 3 to 8 were pooled and diluted to 1000 ml with 50 mM HEPES pH7.0 before application on a Q Sepharose® Fast Flow column XK26/20 60 mlcolumn (GE Healthcare, HiHerod, Denmark). After application the columnwas washed 3 times with 60 ml of 50 mM HEPES pH 7.0 and eluded with 100ml of 50 mM HEPES pH 7.0, 1 M NaCl at a flow rate of 10 ml/minute.Fractions of 10 ml were collected and analyzed by SDS-PAGE. Theflow-through and first wash were pooled and concentrated to 400 ml andwashed with 50 mM HEPES pH 7.0 using the ultrafiltration systemdescribed above. Further concentration was conducted using a VIVASPIN™centrifugal concentrator according to the manufacturer's instructions toa final volume of 80 ml. The protein concentration was determined byA280/A260 absorbance. Protein concentration was also determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 66: Preparation of Neosartorya fischeri Strain NRRL 181 GH7Cellobiohydrolase I

The Neosartorya fischeri NRRL 181 GH7 cellobiohydrolase I (SEQ ID NO:163 [DNA sequence] and SEQ ID NO: 164 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the cellobiohydrolase I gene from Neosartorya fischeri genomicDNA. Genomic DNA was isolated using a FastDNA Spin for Soil Kit.

Primer #374: (SEQ ID NO: 213) 5′-TAAGAATTCACCATGCCTTCCACCTACGA-3′Primer #375: (SEQ ID NO: 214) 5′-TATGCGGCCGCATTCTCCTAGACACCCCGCAT-3′

The amplification reaction was composed of 1 μl of Neosartorya fischerigenomic DNA, 12.5 μl of 2× REDDYMIX™ PCR Buffer, 1 μl of 5 μM primer#374, 1 μl of 5 μM primer #375, and 9.5 μl of H2O. The amplificationreaction was incubated in a PTC-200 DNA ENGINE™ Thermal Cyclerprogrammed for 1 cycle at 94° C. for 2 minutes; and 35 cycles each at94° C. for 15 seconds and 60° C. for 1.5 minutes.

A 1.6 kb PCR reaction product was isolated by 1% agarose gelelectrophoresis using TAE buffer and staining with SYBR Safe DNA gelstain. The DNA band was visualized with the aid of an Eagle Eye ImagingSystem and a DarkReader Transilluminator. The 1.6 kb DNA band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 1.6 kb fragment was cleaved with EcoR I and Not I and purified usinga GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The cleaved 1.6 kb fragment was then directionally cloned by ligationinto Eco RI-Not I cleaved pXYG1051 (WO 2005/080559) using T4 ligaseaccording to the manufacturer's instructions. The ligation mixture wastransformed into E. coli TOP10F competent cells according to themanufacturer's instructions. The transformation mixture was plated ontoLB plates supplemented with 100 μg of ampicillin per ml. Plasmidminipreps were prepared from several transformants and sequenced. Oneplasmid with the correct Neosartorya fischeri GH7 cellobiohydrolasecoding sequence was chosen. The plasmid was designated pXYG1051-NP003786.

The expression plasmid pXYG1051-NP003786 was transformed intoAspergillus oryzae JaL355 as described in WO 98/00529. Transformantswere purified on selection plates to single conidia prior to sporulatingthem on PDA plates. Production of the Neosartorya fischeri GH7cellobiohydrolase by the transformants was analyzed from culturesupernatants of 1 ml 96 deep well stationary cultivations at 26° C. inYP medium with 2% maltodextrin. Expression was verified by SDS-PAGEusing a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer and Coomassieblue staining. One transformant was selected for further work anddesignated Aspergillus oryzae 92.7.

For larger scale production, Aspergillus oryzae 92.7 spores were spreadonto a PDA plate and incubated for five days at 37° C. The confluentspore plate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspension was then used toinoculate twenty-five 500 ml flasks containing 100 ml of YPM medium. Theculture was incubated at 26° C. with constant shaking at 120 rpm. At dayfive post-inoculation, the culture broth was collected by filtrationthrough a triple layer of Whatman glass microfiber filters of 1.6 μm,1.2 μm, and 0.7 μm. Fresh culture broth from this transformant produceda band of GH7 protein of approximately 70 kDa. The identity of this bandas the Neosartorya fischeri GH7 cellobiohydrolase was verified bypeptide sequencing.

Two liters of the filtered broth were concentrated to 400 ml and washedwith 50 mM HEPES pH 7.0 using a SARTOFLOW® Alpha ultrafiltration systemwith a 10 kDa MW-CO. Ammonium sulphate was added to a finalconcentration of 1 M and dissolved in the ultrafiltrate. The solutionwas applied to a Source 15 Phenyl XK 26/20 50 ml column. Afterapplication the column was washed with 150 ml of 1 M ammonium sulphateand eluded with 1 column volume of 50% ethanol in a 0% to 100% gradientfollowed by 5 column volumes of 50% ethanol at a flow rate of 10ml/minute. Fractions of 10 ml were collected and analyzed by SDS-PAGE.Fraction 3 to 8 were pooled and diluted to 1000 ml with 50 mM HEPES pH7.0 before application to a Q Sepharose® Fast Flow XK26/20 60 ml column.After application the column was washed 3 times with 60 ml of 50 mMHEPES pH 7.0 and eluded with 100 ml of 50 mM HEPES pH 7.0, 1 M NaCl at aflow rate of 10 ml/minute. Fractions of 10 ml were collected andanalyzed by SDS-PAGE. The flow-through and first wash were pooled andconcentrated to 400 ml and washed with 50 mM HEPES pH 7.0 using aSARTOFLOW® Alpha ultrafiltration system with a 10 kDa MW-CO. Furtherconcentration was conducted using a VIVASPIN™ centrifugal concentratoraccording to the manufacturer's instructions to a final volume of 80 ml.The protein concentration determined by A280/A260 absorbance. Proteinconcentration was also determined using a Microplate BCA™ Protein AssayKit in which bovine serum albumin was used as a protein standard.

Example 67: Preparation of Aspergillus nidulans Strain FGSCA4 GH7Cellobiohydrolase I

The Aspergillus nidulans strain FGSCA4 GH7 cellobiohydrolase I (SEQ IDNO: 165 [DNA sequence] and SEQ ID NO: 166 [deduced amino acid sequence])was obtained according to the procedure described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the cellobiohydrolase I gene from Aspergillus nidulans genomicDNA. Genomic DNA was isolated using a FastDNA Spin for Soil Kit.

Primer #376: (SEQ ID NO: 215) 5′-TAACAATTGACCATGGCATCTTCATTCCAGTTGTA-3′Primer #377: (SEQ ID NO: 216) 5′-TATGCGGCCGCGTCTCCCATTTACGACCCACCA-3′

The amplification reaction was composed of 1 μl of Aspergillus nidulansgenomic DNA, 12.5 μl of 2× REDDYMIX™ PCR Buffer, 1 μl of 5 μM primer#374, 1 μl of 5 μM primer #375, and 9.5 μl of H₂O. The amplificationreaction was incubated in a PTC-200 DNA ENGINE™ Thermal Cyclerprogrammed for 1 cycle at 94° C. for 2 minutes; and 35 cycles each at94° C. for 15 seconds and 60° C. for 1.5 minutes.

A 1.6 kb PCR reaction product was isolated by 1% agarose gelelectrophoresis using TAE buffer and staining with SYBR Safe DNA gelstain. The DNA band was visualized with the aid of an Eagle Eye ImagingSystem and a DarkReader Transilluminator. The 1.6 kb DNA band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 1.6 kb fragment was cleaved with Mfe I and Not I and purified usinga GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The cleaved 1.6 kb fragment was then directionally cloned by ligationinto Eco RI-Not I cleaved pXYG1051 (WO 2005/080559) using T4 ligaseaccording to the manufacturer's instructions. The ligation mixture wastransformed into E. coli TOP10F competent cells according to themanufacturer's instructions. The transformation mixture was plated ontoLB plates supplemented with 100 μg of ampicillin per ml. Plasmidminipreps were prepared from several transformants and sequenced. Oneplasmid with the Aspergillus nidulans GH7 coding sequence was chosen.Two mutations introduced during PCR were identified which result in achange of Leu 7 to Trp and of Glu 436 to Gly relative to the publicsequence Q8NK02. The plasmid was designated pXYG1051-NP003787.

The expression plasmid pXYG1051-NP003787 was transformed intoAspergillus oryzae JaL355 as described in WO 98/00529. Transformantswere purified on selection plates to single conidia prior to sporulatingthem on PDA plates. Production of the Aspergillus nidulans GH7cellobiohydrolase by the transformants was analyzed from culturesupernatants of 1 ml 96 deep well stationary cultivations at 26° C. inYP medium with 2% maltodextrin. Expression was verified by SDS-PAGEusing a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer and Coomassieblue staining. One transformant was selected for further work anddesignated Aspergillus oryzae 70.5.

For larger scale production, Aspergillus oryzae 70.5 spores were spreadonto a PDA plate and incubated for five days at 37° C. The confluentspore plate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspension was then used toinoculate twenty-five 500 ml flasks containing 100 ml of YPM medium. Theculture was incubated at 26° C. with constant shaking at 120 rpm. At daysix post-inoculation, the culture broth was collected by filtrationthrough a triple layer of Whatman glass microfiber filters of 1.6 μm,1.2 μm, and 0.7 μm. Fresh culture broth from this transformant produceda band of GH7 protein of approximately 54 kDa. The identity of this bandas the Aspergillus nidulans GH7 cellobiohydrolase was verified bypeptide sequencing.

Two liters of the filtered broth were concentrated to 400 ml and washedwith 50 mM HEPES pH 7.0 using a SARTOFLOW® Alpha ultrafiltration systemwith a 10 kDa MW-CO. Ammonium sulphate was added to a finalconcentration of 1 M and dissolved in the ultrafiltrate. The solutionwas applied on a Source 15 Phenyl XK 26/20 50 ml column. Afterapplication the column was washed with 150 ml of 1 M ammonium sulphateand eluded with 1 column volume of 50% ethanol in a 0% to 100% gradientfollowed by 5 column volumes of 50% ethanol at a flow rate of 10ml/minute. Fractions of 10 ml were collected and analyzed by SDS-PAGE.Fraction 3 to 8 were pooled and diluted to 1000 ml with 50 mM HEPES pH7.0 before application on a Q Sepharose® Fast Flow XK26/20 60 ml column.After application the column was washed 3 times with 60 ml of 50 mMHEPES pH 7.0 and eluded with 100 ml of 50 mM HEPES pH 7.0, 1 M NaCl at aflow rate of 10 ml/minute. Fractions of 10 ml were collected andanalyzed by SDS-PAGE. The flow-through and first wash were pooled andconcentrated to 400 ml and washed with 50 mM HEPES pH 7.0 using aSARTOFLOW® Alpha ultrafiltration system with a 10 kDa MW-CO. Furtherconcentration was conducted using a VIVASPIN™ centrifugal concentratoraccording to the manufacturer's instructions to a final volume of 80 ml.The protein concentration determined by A280/A260 absorbance. Proteinconcentration was also determined using a Microplate BCA™ Protein AssayKit in which bovine serum albumin was used as a protein standard.

Example 68: Preparation of a Fennellia nivea Strain NN046949 GH6Cellobiohydrolase II

The Fennellia nivea strain NN046949 GH6 cellobiohydrolase II (SEQ ID NO:167 [DNA sequence] and SEQ ID NO: 168 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Fennellia nivea strain NN046949 was isolated from a soil from Yunnan,China by directly plating the soil sample onto a PDA plate followed byincubation at 37° C. for 5 days. The strain was then purified bytransferring the mycelia onto a YG agar plate. The Fennellia niveastrain NN046949 was identified as Fennellia nivea based on bothmorphological and molecular (ITS sequencing) characterization.

Fennellia nivea strain NN046949 was inoculated onto a PDA plate andincubated for 4 days at 37° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml ofNNCYP-PCS medium. The flasks were incubated for 6 days at 37° C. withshaking at 160 rpm. The mycelia were collected at 4, 5, and 6 days, andeach frozen in liquid nitrogen and stored in a −80° C. freezer untiluse.

The frozen F. nivea mycelia were mixed and transferred into a liquidnitrogen prechilled mortar and pestle and ground to a fine powder. TotalRNA was prepared from the powdered mycelia of each day by extractionwith TRIZOL® reagent and purified using a RNEASY® Plant Mini Kitaccording to the manufacturer's protocol. The polyA enriched RNA wasisolated using a mTRAP™ Total Kit. Eighty-seven μs of total RNA wassubmitted for sequencing as described in Example 3.

Total RNA enriched for polyA sequences with the mRNASeq protocol weresequenced using an ILLUMINA® GA2 System (Illumina, Inc., San Diego,Calif., USA). The raw 75 base pair reads were assembled and theassembled sequences were analyzed using standard bioinformatics methodsfor gene finding and functional prediction. Briefly, ESTscan 2.0 wasused for gene prediction. NCBI blastaII version 2.2.10 and HMMER version2.1.1 were used to predict function based on structural homology. TheFamily GH6 cellobiohydrolase was identified directly by analysis of theBlast results.

Fennellia nivea strain NN046949 was grown on PDA agar plate at 37° C.for 3 days. Mycelia were collected directly from the agar plate into asterilized motar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Based on the F. nivea GH6 cellobiohydrolase gene sequence obtained inExample 3, oligonucleotide primers, shown below, were designed toamplify the gene from genomic DNA of Fennellia nivea. An IN-FUSION™ CFDry-down PCR Cloning Kit was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 217)5′-ACACAACTGGGGATCCACCATGGGACGGGTTTCTTCTCTTG-3′ Antisense primer:(SEQ ID NO: 218) 5′-GTCACCCTCTAGATCTAAGAACACCCCGCAAAGAAAGTC-3′Bold letters represent the coding sequence for the sense primer or thereverse compliment sequence downstream of the stop codon for theantisense primer. The remaining sequence is homologous to the insertionsites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Fennellia nivea genomic DNA, 10 μl of 5×GC Buffer,1.5 μl of DMSO, 2 μl of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 1unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of 50μl. The amplification was performed using an Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minutes; 5 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 70° C. for 30 seconds,with 1° C. decreasing per cycle and elongation at 72° C. for 30 seconds;25 cycles each at 98° C. for 15 seconds and 72° C. for 90 seconds; and afinal extension at 72° C. for 10 minutes. The heat block then went to a4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.8 kb product band was excisedfrom the gel, and purified using an ILLUSTRA™ GFX™ PCR DNA and Gel BandPurification Kit (GE Healthcare, Buckinghamshire, UK) according to themanufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions. The gene fragment and the digested vectorwere ligated together using an IN-FUSION™ Dry Down PCR Cloning Kitresulting in pCBHII46949-2 in which transcription of the Fennellia niveaGH6 cellobiohydrolase gene was under the control of the Aspergillusoryzae TAKA alpha-amylase promoter. In brief, 30 ng of pPFJO355 digestedwith Barn I and Bgl II, and 50 ng of the F. nivea GH6 cellobiohydrolasegene purified PCR product were added to a reaction vial and resuspendedin a final volume of 10 μl with addition of deionized water. Thereaction was incubated at 37° C. for 15 minutes and then 50° C. for 15minutes. Three μl of the reaction were used to transform E. coli TOP10competent cells. An E. coli transformant containing pCBHII46949-2 wasdetected by colony PCR and plasmid DNA was prepared using a QIAprep SpinMiniprep Kit (QIAGEN Inc., Valencia, Calif., USA). The F. nivea GH6cellobiohydrolase gene insert in pCBHII46949-2 was confirmed by DNAsequencing using a 3730XL DNA Analyzer.

The same gene fragment was then incubated in 10×Taq DNA polymerase mix(TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China) at 72° C. for 20minutes to add adenine to the 3′ end of each nucleotide strand. Then thegene fragment was ligated to pGEM-T vector using a pGEM-T Vector Systemto generate pGEM-T-CBHII46949-2. The Fennellia nivea cellobiohydrolasegene insert in pGEM-T-CBHII46949-2 was confirmed by DNA sequencing usinga 3730XL DNA Analyzer. E. coli strain T-CBHII46949-2, containingpGEM-T-CBHII46949-2, was deposited on Oct. 28, 2010 with the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), MascheroderWeg 1 B, D-38124 Braunschweig, Germany assigned the accession number DSM24143.

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of the F. niveacellobiohydrolase gene are shown in SEQ ID NO: 167 and SEQ ID NO: 168,respectively. The genomic fragment encodes a polypeptide of 469 aminoacids, interrupted by 7 predicted introns of 49 bp (nucleotides 77-125),247 bp (nucleotides 195-241), 46 bp (nucleotides 570-615), 55 bp(nucleotides 870-924), 50 bp (nucleotides 1063-1112), 46 bp (nucleotides1371-1416), and 49 bp (nucleotides 1659-1707). The % G+C content of thefull-length coding sequence and the mature coding sequence are 57.65%and 60.24%, respectively. Using the SignalP software program (Nielsen etal., 1997, Protein Engineering 10: 1-6), a signal peptide of 18 residueswas predicted. The predicted mature protein contains 451 amino acidswith a predicted molecular mass of 48.77 kDa and an isoelectric point of5.17. Amino acids 112 to 469 are indicative of a Family 6 glycosylhydrolase. Based on the deduced amino acid sequence, thecellobiohydrolase appears to fall into the cellobiohydrolase Family GH6according to Coutinho and Henrissat, 1999, supra. Amino acids 22 to 50are indicative of a CBM domain and amino acids 58 to 111 a linkerregion.

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pCBHII46949-2. The transformationyielded about 50 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium in24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation,20 μl of supernatant from each culture were analyzed by SDS-PAGE using aNUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer according to themanufacturer's instructions. The resulting gel was stained withINSTANTBLUE™ (Expedeon Ltd., Babraham Cambridge, UK). SDS-PAGE profilesof the cultures showed that the majority of the transformants had a bandof approximately 60 kDa. One transformant was chosen as the expressionstrain and designated Aspergillus oryzae EXP03324.

A slant of Aspergillus oryzae EXP03324 was washed with 10 ml of YPMmedium and inoculated into 4 2-liter flasks, containing 400 ml of YPMmedium for each, to generate broth for characterization of the enzyme.The culture was harvested on day 3 by filtering the culture againstMIRACLOTH® (CALBIOCHEM, Inc. La Jolla, Calif., USA). The filteredculture broth was then again filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

A 1600 ml volume of the Aspergillus oryzae EXP03324 filtered broth wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml 25 mM Bis-Tris pH 6.5 buffer, dialyzed against the same buffer, andfiltered through a 0.45 μm filter; the final volume was 200 ml. Thesolution was applied to a 40 ml Q Sepharose® Fast Flow columnequilibrated in 25 mM Bis-Tris pH 6.5 buffer and the proteins wereeluted with a linear 0-0.4 M NaCl gradient. Fractions from the columnwere analyzed by SDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel withMES buffer. Fractions with a molecular weight of 60 kDa were pooled.Then the pooled solution was concentrated by ultrafiltration and assayedfor cellobiohydrolase activity using phosphoric acid swollen cellulose(PASC) as substrate. Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

A PASC stock slurry solution was prepared by moistening 5 g of AVICEL®(JRS Pharma GmbH & Co., Rosenberg, Germany) with water, followed by theaddition of 150 ml of ice cold 85% o-phosphoric acid. The suspension wasslowly stirred in an ice-bath for 1 hour. Then 500 ml of ice coldacetone were added while stirring. The slurry was filtered usingMIRACLOTH® and then washed three times with 100 ml of ice-cold acetone(drained as dry as possible after each wash). Finally, the filteredslurry was washed twice with 500 ml of water, and again drained as dryas possible after each wash. The PASO was mixed with deionized water toa total volume of 500 ml to a concentration of 10 g/liter, blended tohomogeneity using an ULTRA-TURRAX® Homogenizer (Cole-Parmer, VernonHills, Ill., USA), and stored in a refrigerator for up to one month.

The PASO stock solution was diluted with 50 mM sodium acetate pH 5.0buffer to a concentration of 2 g/liter, and used as the substrate. To150 μl of PASO stock solution, 20 μl of enzyme sample were added and thereaction mixture was incubated for 60 minutes with shaking at 850 rpm.At the end of the incubation, 50 μl of 2% NaOH were added to stop thereaction. The reaction mixture was centrifuged at 1,000×g. The releasedsugars were measured by first mixing 10 μl of the reaction mixture with90 μl of 0.4% NaOH, followed by 50 μl of 1.5% p-hydroxybenzoic acidhydrazide in 2% NaOH (PHBAH, Sigma Chemical Co., St. Louis, Mo., USA).The mixture was boiled at 100° C. for 5 minutes, and then 100 μl weretransferred to a microtiter plate for an absorbance reading at 410 nmusing a Spectra Max M2 (Molecular Devices, Sunnyvale, Calif., USA).Blanks were made by omitting PASO in the hydrolysis step, and byreplacing the hydrolysate with buffer in the sugar determination step.

The assay results demonstrated that the purified enzyme possessedcellobiohydrolase activity.

Example 69: Preparation of Penicillium emersonii Strain NN051602 GH6ACellobiohydrolase II

Penicillium emersonii strain NN051602 GH6A cellobiohydrolase II (SEQ IDNO: 169 [DNA sequence] and SEQ ID NO: 170 [deduced amino acid sequence])was obtained according to the procedure described below.

Penicillium emersonii was grown on a PDA agar plate at 45° C. for 3days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Oligonucleotide primers, shown below, were designed to amplify the GH6cellobiohydrolase II gene from genomic DNA of Penicillium emersonii. AnIN-FUSION™ CF Dry-down Cloning Kit was used to clone the fragmentdirectly into the expression vector pPFJO355, without the need forrestriction digestion and ligation.

Sense primer: (SEQ ID NO: 219)5′-ACACAACTGGGGATCCACCATGCGGAATCTTCTTGCTCTTGC-3′ Antisense primer:(SEQ ID NO: 220) 5′-GTCACCCTCTAGATCTCTAGAACAGCGGGTTAGCATTCGTG-3′Bold letters represented the coding sequence. The remaining sequence washomologous to the insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium emersonii genomic DNA, 10 μl of 5×HFBuffer, 1.5 μl of DMSO, 5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of 50μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minutes; 8 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 66° C. for 30 seconds,with a 1° C. decrease per cycle and elongation at 72° C. for 70 seconds;and another 25 cycles each at 98° C. for 15 seconds, 62° C. for 30seconds and 72° C. for 80 seconds; final extension at 72° C. for 5minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.8 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in pCBHII51602 in whichtranscription of the Penicillium emersonii GH6 cellobiohydrolase II genewas under the control of the Aspergillus oryzae TAKA amylase promoter.In brief, 30 ng of pPFJO355, digested with Barn I and Bgl II, and 60 ngof the Penicillium emersonii GH6 cellobiohydrolase II gene purified PCRproduct were added to a reaction vial and resuspended in a final volumeof 10 μl with addition of deionized water. The reaction was incubated at37° C. for 15 minutes and then 50° C. for 15 minutes. Three μl of thereaction were used to transform E. coli TOP10 competent cells. An E.coli transformant containing pCBHII51602 was detected by colony PCR andplasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc.,Valencia, Calif., USA). The Penicillium emersonii GH6 cellobiohydrolaseII gene insert in pCBHII51602 was confirmed by DNA sequencing using a3730XL DNA Analyzer.

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pCBHII51602. The transformationyielded about 50 transformants. Twelve transformants were isolated toindividual Minimal medium plates.

Six transformants were inoculated separately into 3 ml of YPM medium in24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation,20 μl of supernatant from each culture were analyzed by SDS-PAGE using aNUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer according to themanufacturer's instructions. The resulting gel was stained with INSTANT®Blue. SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major smeary band of approximately 62 kDa. Theexpression strain was designated as A. oryzae EXP03259.

A slant of A. oryzae EXP03259 was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane.

A 1600 ml volume of filtered broth of A. oryzae EXP03259 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml 2 of 5 mM Bis-Tris pH 6.0, dialyzed against the same buffer, andfiltered through a 0.45 μm filter; the final volume was 200 ml. Thesolution was applied to a 40 ml Q Sepharose® Fast Flow columnequilibrated with 25 mM Bis-Tris pH 6.0, and the proteins were elutedwith a linear NaCl gradient (0-0.4 M). Fractions from the column withactivity against PASC were analyzed by SDS-PAGE using a NUPAGE® NOVEX®4-12% Bis-Tris Gel with MES buffer. Fractions with the correct molecularweight were pooled and concentrated by ultrafiltration. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 70: Preparation of Penicillium pinophilum Strain NN046877 GH6ACellobiohydrolase II

The Penicillium pinophilum strain NN046877 GH6A cellobiohydrolase II(SEQ ID NO: 171 [DNA sequence] and SEQ ID NO: 172 [deduced amino acidsequence]) was obtained according to the procedure described below.

Penicillium pinophilum was grown on a PDA agar plate at 37° C. for 4-5days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Mini Kit (QIAGEN Inc., Valencia, Calif.,USA).

Oligonucleotide primers, shown below, were designed to amplify the GH6cellobiohydrolase II gene from genomic DNA of Penicillium pinophilum. AnIN-FUSION™ CF Dry-down Cloning Kit was used to clone the fragmentdirectly into the expression vector pPFJO355, without the need forrestriction digestion and ligation.

Sense primer: (SEQ ID NO: 221)5′-ACACAACTGGGGATCCACCATGTTGCGATATCTTTCCACC-3′ Antisense primer:(SEQ ID NO: 222) 5′-GTCACCCTCTAGATCTTCATCTAGACCAAAGCTGGGTTG-3′Bold letters represented the coding sequence and the remaining sequencewas homologous to the insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Penicillium pinophilum genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and0.6 unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of50 μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 1 minutes; 5 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 56° C. for 30 seconds,with a 1° C. increase per cycle and elongation at 72° C. for 75 seconds;and another 25 cycles each at 98° C. for 15 seconds, 65C for 30 secondsand 72° C. for 75 seconds; final extension at 72° C. for 10 minutes. Theheat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.7 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in pPpin12 in whichtranscription of the Penicillium pinophilum GH6 cellobiohydrolase IIgene was under the control of the Aspergillus oryzae TAKA amylasepromoter. In brief, 30 ng of pPFJO355, digested with Barn I and Bgl II,and 60 ng of the Penicillium pinophilum GH6 cellobiohydrolase II genepurified PCR product were added to a reaction vial and resuspended in afinal volume of 10 ul with addition of deionized water. The reaction wasincubated at 37° C. for 15 minutes and then 50° C. for 15 minutes. Threeμl of the reaction were used to transform E. coli TOP10 competent cells.An E. coli transformant containing pPpin12 was detected by colony PCRand plasmid DNA was prepared using a QIAprep Spin Miniprep Kit. ThePenicillium pinophilum GH6 cellobiohydrolase II gene insert in pPpin12was confirmed by DNA sequencing using a 3730XL DNA Analyzer.

Aspergillus oryzae HowB101 (WO9535385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pPpin12. The transformationyielded about 50 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium in24-well plate and incubated at 30° C., 150 rpm. After 3 days incubation,20 μl of supernatant from each culture were analyzed by SDS-PAGE using aNUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES buffer according to themanufacturer's instructions. The resulting gel was stained with INSTANT®Blue. SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 65 kDa. The expressionstrain was designated A. oryzae EXP02774.

A slant of A. oryzae EXP02774 was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane.

A 1600 ml volume of the filtered broth of A. oryzae EXP02774 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml 25 mM Bis-Tris pH 6.0, dialyzed against the same buffer, and filteredthrough a 0.45 μm filter; the final volume was 200 ml. The solution wasapplied to a 40 ml Q Sepharose® Fast Flow column equilibrated in 25 mMBis-Tris pH 6.0, and the proteins were eluted with a linear NaClgradient (0-0.4 M). Fractions from the column with activity against PASOwere collected and applied to a 40 ml HITRAP® SP Fast Flow columnequilibrated in 25 mM Bis-Tris pH 6.0, and the proteins were eluted witha linear NaCl gradient (0-0.4 M). Fractions from the column withactivity against PASO were analyzed by SDS-PAGE using a NUPAGE® NOVEX®4-12% Bis-Tris Gel with MES buffer. Fractions with the correct molecularweight were pooled and concentrated by ultrafiltration. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 71: Preparation of Aspergillus fumigatus Strain NN051616 GH5Endoglucanase II

The Aspergillus fumigatus strain NN051616 GH5 endoglucanase II (SEQ IDNO: 173 [DNA sequence] and SEQ ID NO: 174 [deduced amino acid sequence])was obtained according to the procedure described below.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Aspergillus fumigatus Family GH5 gene from the genomic DNA.An IN-FUSION™ Cloning Kit was used to clone the fragment directly intothe expression vector, pAILo2 (WO 2005/074647), without the need forrestriction digests and ligation.

Forward primer: (SEQ ID NO: 223)5′-ACTGGATTTACCATGAAATTCGGTAGCATTGTGCTC-3′ Reverse primer:(SEQ ID NO: 224) 5′-TCACCTCTAGTTAATTAATCAACCCAGGTAGGGCTCCAAGATG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifteen picomoles of each of the primers above were used in a PCRreaction containing 200 ng of Aspergillus fumigatus genomic DNA, 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 1 mM MgSO₄,1.5 μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PfxDNA polymerase (Invitrogen, Carlsbad, Calif., USA), in a final volume of50 μl. The amplification conditions were one cycle at 98° C. for 3minutes; and 30 cycles each at 98° C. for 30 seconds, 57° C. for 30seconds, and 72° C. for 75 seconds. The heat block was then held at 72°C. for 15 minutes followed by a 4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer and a 2.4 kb product band was excised from the gel and purifiedusing a MinElute® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.)according to the manufacturer's instructions. The fragment was thencloned into pAILo2 using an IN-FUSION™ Cloning Kit. The vector wasdigested with Nco I and Pac I. The fragment was purified by gelelectrophoresis and a QlAquick Kit (QIAGEN Inc., Valencia, Calif., USA).The gene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG10, in whichtranscription of the Aspergillus fumigatus Family GH5 gene was under thecontrol of the NA2-tpi promoter (a hybrid of the promoters from thegenes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase). The recombination reaction (20 μl) wascomposed of 1×I IN-FUSION™ Buffer (Clontech, Mountain View, Calif.),1×BSA (Clontech, Mountain View, Calif.), 1 μl of IN-FUSION™ enzyme(diluted 1:10) (Clontech, Mountain View, Calif.), 180 ng of pAILo2digested with Nco I and Pac I, and 80 ng of the Aspergillus fumigatusbeta-xylosidase purified PCR product. The reaction was incubated atambient temperature for 30 minutes. The reaction was diluted with 40 μlof TE buffer and 2.5 μl of the diluted reaction was used to transform E.coli SOLOPACK® Gold cells. An E. coli transformant containing pAG10(Aspergillus fumigatus Family GH5 gene) was identified by restrictionenzyme digestion and plasmid DNA was prepared using a QIAGEN BioRobot9600. The pAG10 plasmid construct was sequenced using an AppliedBiosystems 3130x1 Genetic Analyzer (Applied Biosystems, Foster City,Calif., USA) to verify the sequence.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422 andtransformed with 5 μg of pAG10. Three transformants were isolated toindividual PDA plates.

Plugs taken from the original transformation plate of each of the threetransformants were added to 1 ml of M410 medium separately in 24 wellplates, which were incubated at 34° C. After five days of incubation,7.5 μl of supernatant from each culture was analyzed using CRITERION®stain-free, 8-16% gradient SDS-PAGE, (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) according to the manufacturer's instructions.SDS-PAGE profiles of the cultures showed that the transformants had anew major band of approximately 35 kDa.

Confluent PDA plate of the highest expressing transformant was washedwith 5 ml of 0.01% TWEEN® 20 and inoculated into three 500 ml Erlenmeyerflasks, each containing 100 ml of M410 medium. Inoculated flasks wereincubated with shaking for 3 days at 34° C. The broths were pooled andfiltered through a 0.22 μm stericup suction filter (Millipore, Bedford,Mass., USA).

A 35 ml volume of filtered broth was buffer exchanged into 50 mM sodiumacetate pH 5.0 using a 400 ml Sephadex G-25 column (GE Healthcare,United Kingdom) according to the manufacturer's instructions. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 72: Preparation of Neosartorya fischeri Strain NRRL 181 GH5Endoglucanase II

The Neosartorya fischeri NRRL 181 GH5 endoglucanase II (SEQ ID NO: 175[DNA sequence] and SEQ ID NO: 176 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the endoglucanase gene from Neosartorya fischeri NRRL 181genomic DNA. Genomic DNA was isolated using a FastDNA spin for Soil Kit.

Primer #350: (SEQ ID NO: 225)5′-TAAGAATTCACCATGAAGGCTTCGACTATTATCTGTGCA-3′ Primer #358:(SEQ ID NO: 226) 5′-TATGCGGCCGCACGGCAATCCAAGTCATTCAA-3′

The amplification reaction was composed of 1 μl of Neosartorya fischerigenomic DNA, 12.5 μl of 2× REDDYMIX™ PCR Buffer, 1 μl of 5 μM primer#374, 1 μl of 5 μM primer #375, and 9.5 μl of H₂O. The amplificationreaction was incubated in a PTC-200 DNA ENGINE™ Thermal Cyclerprogrammed for 1 cycle at 94° C. for 2 minutes; and 35 cycles each at94° C. for 15 seconds and 60° C. for 1.5 minutes.

A 1.4 kb PCR reaction product was isolated by 1% agarose gelelectrophoresis using TAE buffer and staining with SYBR Safe DNA gelstain. The DNA band was visualized with the aid of an Eagle Eye ImagingSystem and a DarkReader Transilluminator. The 1.4 kb DNA band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

The 1.4 kb fragment was cleaved with EcoR I and Not I and purified usinga GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The cleaved 1.4 kb fragment was then directionally cloned by ligationinto Eco RI-Not I cleaved pXYG1051 (WO 2005/080559) using T4 ligaseaccording to the manufacturer's instructions. The ligation mixture wastransformed into E. coli TOP10F competent cells according to themanufacturer's instructions. The transformation mixture was plated ontoLB plates supplemented with 100 μg of ampicillin per ml. Plasmidminipreps were prepared from several transformants and sequenced. Oneplasmid with the correct Neosartorya fischeri GH5 coding sequence waschosen.

The expression plasmid pXYG1051-NP003772 was transformed intoAspergillus oryzae JaL355 as described in WO 98/00529. Transformantswere purified on selection plates to single conidia prior to sporulatingthem on PDA plates. Production of the Neosartorya fischeri GH5polypeptide by the transformants was analyzed from culture supernatantsof 1 ml 96 deep well stationary cultivations at 26° C. in YP medium with2% maltodextrin. Expression was verified SDS-PAGE using a NUPAGE® NOVEX®4-12% Bis-Tris Gel with MES buffer and Coomassie blue staining. Onetransformant was selected for further work and designated Aspergillusoryzae 83.3.

For larger scale production, Aspergillus oryzae 83.3 spores were spreadonto a PDA plate and incubated for five days at 37° C. The confluentspore plate was washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspension was then used toinoculate twenty-five 500 ml flasks containing 100 ml of YPG medium. Theculture was incubated at 26° C. with constant shaking at 120 rpm. At dayfive post-inoculation, the culture broth was collected by filtrationthrough a triple layer of Whatman glass microfiber filters of 1.6 μm,1.2 μm, and 0.7 μm. Fresh culture broth from this transformant produceda band of GH7 protein of approximately 46 kDa. The identity of this bandas the Neosartorya fischeri GH5 polypeptide was verified by peptidesequencing.

Two liters of the filtered broth were concentrated to 400 ml and washedwith 50 mM HEPES pH 7.0 using a SARTOFLOW® Alpha ultrafiltration systemwith a 10 kDa MW-CO. Ammonium sulphate was added to a finalconcentration of 1 M and dissolved in the ultrafiltrate. The solutionwas applied on a Source 15 Phenyl XK 26/20 50 ml column. Afterapplication the column was washed with 150 ml of 1 M ammonium sulphateand eluded with 1 column volume of 50% ethanol in a 0% to 100% gradientfollowed by 5 column volumes of 50% ethanol at a flow rate of 10ml/minute. Fractions of 10 ml were collected and analyzed by SDS-PAGE.Fraction 3 to 8 were pooled and diluted to 1000 ml with 50 mM HEPES pH7.0 before application on a Q Sepharose® Fast Flow column XK26/20 60 mlcolumn. After application the column was washed 3 times with 60 ml of 50mM HEPES pH 7.0 and eluded with 100 ml of 50 mM HEPES pH 7.0, 1 M NaClat a flow rate of 10 ml/minute. Fractions of 10 ml were collected andanalyzed by SDS-PAGE. The flow-through and first wash were pooled andconcentrated to 400 ml and washed with 50 mM HEPES pH 7.0 using theultrafiltration system described above. Further concentration wasconducted using a VIVASPIN™ centrifugal concentrator according to themanufacturer's instructions to a final volume of 80 ml. The proteinconcentration was determined by A280/A260 absorbance. Proteinconcentration was also determined using a Microplate BCA™ Protein AssayKit in which bovine serum albumin was used as a protein standard.

Example 73: Preparation of Aspergillus aculeatus Strain WDCM190 GH3Beta-Glucosidase

The Aspergillus aculeatus strain WDCM190 GH3 beta-glucosidase (SEQ IDNO: 177 [DNA sequence] and SEQ ID NO: 178 [deduced amino acid sequence])was obtained according to the procedure described below.

To generate genomic DNA for PCR amplification, Aspergillus aculeatusWDCM 190 was propagated on PDA agar plates by growing at 26° C. for 7days. Spores harvested from the PDA plates were inoculated into 25 ml ofYP+2% glucose medium in a baffled shake flask and incubated at 26° C.for 48 hours with agitation at 200 rpm.

Genomic DNA was isolated according to a modified FastDNA® SPIN protocol(Qbiogene, Inc., Carlsbad, Calif., USA). Briefly, a FastDNA® SPIN Kitfor Soil (Qbiogene, Inc., Carlsbad, Calif., USA) was used in a FastPrep®24 Homogenization System (MP Biosciences, Santa Ana, Calif., USA). Twoml of fungal material were harvested by centrifugation at 14,000×g for 2minutes. The supernatant was removed and the pellet resuspended in 500μl of deionized water. The suspension was transferred to a Lysing MatrixE FastPrep® tube (Qbiogene, Inc., Carlsbad, Calif., USA) and 790 μl ofsodium phosphate buffer and 100 μl of MT buffer from the FastDNA® SPINKit were added to the tube. The sample was then secured in a FastPrep®Instrument (Qbiogene, Inc., Carlsbad, Calif., USA) and processed for 60seconds at a speed of 5.5 m/sec. The sample was then centrifuged at14000×g for two minutes and the supernatant transferred to a cleanEPPENDORF® tube. A 250 μl volume of PPS reagent from the FastDNA® SPINKit was added and then the sample was mixed gently by inversion. Thesample was again centrifuged at 14000×g for 5 minutes. The supernatantwas transferred to a 15 ml tube followed by 1 ml of Binding Matrixsuspension from the FastDNA® SPIN Kit and then mixed by inversion fortwo minutes. The sample was placed in a stationary tube rack and thesilica matrix was allowed to settle for 3 minutes. A 500 μl volume ofthe supernatant was removed and discarded and then the remaining samplewas resuspended in the matrix. The sample was then transferred to a SPINfilter tube from the FastDNA® SPIN Kit and centrifuged at 14000×g for 1minute. The catch tube was emptied and the remaining matrix suspensionadded to the SPIN filter tube. The sample was again centrifuged(14000×g, 1 minute). A 500 μl volume of SEWS-M solution from theFastDNA® SPIN Kit was added to the SPIN filter tube and the sample wascentrifuged at the same speed for 1 minute. The catch tube was emptiedand the SPIN filter replaced in the catch tube. The unit was centrifugedat 14000×g for 2 minutes to “dry” the matrix of residual SEWS-M washsolution. The SPIN filter was placed in a fresh catch tube and allowedto air dry for 5 minutes at room temperature. The matrix was gentlyresuspended in 100 μl of DES (DNase/Pyrogen free water) with a pipettetip. The unit was centrifuged (14000×g, 1 minute) to elute the genomicDNA followed by elution with 100 μl of 10 mM Tris, 0.1 mM EDTA, pH 8.0by renewed centrifugation at 14000×g for 1 minute and the eluates werecombined. The concentration of the DNA harvested from the catch tube wasmeasured by a UV spectrophotometer at 260 nm.

The Aspergillus aculeatus Cel3 beta-glucosidase gene was isolated by PCRusing two cloning primers GH3-8f and GH3-8r shown below, which weredesigned based on the publicly available Aspergillus aculeatus Cel3 mRNAsequence (Genbank D64088.1) for direct cloning by IN-FUSION™ strategy.

Primer GH3-8f: (SEQ ID NO: 227)5′-acacaactggggatccaccatgaagctcagttggcttgaggc gg-3′ Primer GH3-8r:(SEQ ID NO: 228) 5′-agatctcgagaagcttattgcaccttcgggagcgccgcgtga ag-3′

A PCR reaction was performed with genomic DNA prepared from Aspergillusaculeatus strain (IAM2445; WDCM190) in order to amplify the full-lengthgene. The PCR reaction was composed of 1 μl of genomic DNA, 0.75 μl ofprimer GH3-8f (10 μM), 0.75 μl of primer GH3-8r (10 μM), 3 μl of 5×HFbuffer, 0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μl ofPHUSION® DNA polymerase, and PCR-grade water up to 15 μl. The PCRreaction was performed using a DYAD® PCR machine programmed for 2minutes at 98° C. followed by 10 touchdown cycles at 98° C. for 15seconds, 70° C. (−1° C./cycle) for 30 seconds, and 72° C. for 2 minutes30 seconds; and 25 cycles each at 98° C. for 15 seconds, 60° C. for 30seconds, 72° C. for 2 minutes 30 seconds, and 5 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Aspergillus aculeatus Cel3 beta-glucosidase genewas cloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an IN-FUSION™ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Aspergillus aculeatusCel3 beta-glucosidase gene sequence was verified by Sanger sequencingbefore heterologous expression.

The coding sequence is 2940 bp including the stop codon and isinterrupted by 6 introns of 73 bp (nucleotides 58 to 130), 52 bp(nucleotides 274 to 325), 57 bp (nucleotides 371 to 427), 61 bp(nucleotides 481 to 541), 64 bp (nucleotides 1734 to 1797), and 50 bp(nucleotides 2657 to 2706). The encoded predicted protein is 860 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 841 amino acids.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000 (Applichem Inc. Omaha, Nebr., USA)(polyethylene glycol, molecular weight 4,000), 10 mM CaCl₂, and 10 mMTris-HCl pH 7.5 were added and gently mixed. The mixture was incubatedat 37° C. for 30 minutes and the protoplasts were spread on COVE sucrose(1 M) plates supplemented with 10 mM acetamide and 15 mM CsCl fortransformant selection. After incubation for 4-7 days at 37° C. sporesof several transformants were seeded on YP-2% maltodextrin medium. After4 days cultivation at 30° C. culture broth was analyzed in order toidentify the best transformants based on their ability to produce alarge amount of active Aspergillus aculeatus Cel3 beta-glucosidase. Thescreening was based on intensity of the band corresponding to theheterologous expressed protein determined by SDS-PAGE and activity ofthe enzyme on 4-nitrophenyl-beta-D-glucopyranoside (pNPG) as describedin Example 16 herein.

Spores of the best transformant designated were spread on COVE platescontaining 0.01% TRITON® X-100 in order to isolate single colonies. Thespreading was repeated twice in total on COVE sucrose medium (Cove,1996, Biochim. Biophys. Acta 133: 51-56) containing 1 M sucrose and 10mM sodium nitrate, supplemented with 10 mM acetamide and 15 mM CsCl.Fermentation was then carried out in 250 ml shake flasks using YP-2%maltodextrin medium for 4 days at 30° C. with shaking at 100 rpm. Thebroth was filtered using standard methods. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 74: Preparation of Aspergillus kawashii Strain IFO 4308 GH3Beta-Glucosidase

The Aspergillus kawashii strain IFO 4308 GH3 beta-glucosidase (SEQ IDNO: 179 [DNA sequence] and SEQ ID NO: 180 [deduced amino acid sequence])was obtained according to the procedure described below.

To generate genomic DNA for PCR amplification, the fungi were propagatedon PDA agar plates by growing at 26° C. for 7 days. Spores harvestedfrom the PDA plates were used to inoculate 25 ml of YP+2% glucose mediumin a baffled shake flask and incubated at 26° C. for 48 hours withagitation at 200 rpm.

Genomic DNA was isolated according to the procedure described in Example73.

The Aspergillus kawachil beta-glucosidase gene was isolated by PCR usingtwo cloning primers GH3-33f and GI-13-33r shown below, which weredesigned based on the publicly available Aspergillus kawachiifull-length sequence (GenBank AB003470.1) for direct cloning byINFUSION™ strategy.

Primer GH3-33f: (SEQ ID NO: 229)acacaactggggatccaccatgaggttcactttgattgaggcgg Primer GH3-33r:(SEQ ID NO: 230) agatctcgagaagcttaGTGAACAGTAGGCAGAGACGCCCGGAGC

A PCR reaction was performed with the genomic DNA prepared fromAspergillus kawachii IFO 4308 in order to amplify the full-length gene.The PCR reaction was composed of 1 μl of genomic DNA, 0.75 μl of primerGH3-33f (10 μM), 0.75 μl of primer GH3-33r (10 μM), 3 μl of 5×HF buffer,0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μl of PHUSION® DNApolymerase, and PCR-grade water up to 15 μl. The PCR reaction wasperformed using a DYAD® PCR machine programmed for 2 minutes at 98° C.followed by 10 touchdown cycles at 98° C. for 15 seconds, 70° C. (−1°C./cycle) for 30 seconds, and 72° C. for 2 minutes 30 seconds; and 25cycles each at 98° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for2 minutes 30 seconds, and 5 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Aspergillus kawachii beta-glucosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an IN-FUSION™′ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Aspergillus kawachiibeta-glucosidase gene sequence was verified by Sanger sequencing beforeheterologous expression.

The coding sequence is 2935 bp including the stop codon and isinterrupted by 6 introns of 92 bp (nucleotides 58 to 149), 48 bp(nucleotides 293 to 340), 54 bp (nucleotides 386 to 439), 51 bp(nucleotides 493 to 543), 57 bp (nucleotides 1736 to 1792), and 50 bp(nucleotides 2652 to 2701). The encoded predicted protein is 860 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 841 amino acids.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 were added and gently mixed. The mixture was incubated at 37° C. for30 minutes and the protoplasts were spread on COVE sucrose (1 M) platessupplemented with 10 mM acetamide and 15 mM CsCl for transformantselection. After incubation for 4-7 days at 37° C. spores of severaltransformants were seeded on YP-2% maltodextrin medium. After 4 dayscultivation at 30° C. culture broth was analyzed in order to identifythe best transformants based on their ability to produce a large amountof active Aspergillus kawachii beta-glucosidase. The screening was basedon intensity of the band corresponding to the heterologous expressedprotein determined by SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-glucopyranoside (pNPG) as described in Example 16herein.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then carried out in 250 ml shake flasks using YP-2% maltodextrinmedium for 4 days at 30° C. with shaking at 100 rpm. The broth wasfiltered using standard methods. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 75: Preparation of Aspergillus clavatus Strain NRRL 1 GH3Beta-Glucosidase

The Aspergillus clavatus strain NRRL 1 GH3 beta-glucosidase (SEQ ID NO:181 [DNA sequence] and SEQ ID NO: 182 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Genomic DNA was isolated according to the procedure described in Example73.

The Aspergillus clavatus beta-glucosidase gene was isolated by PCR usingtwo cloning primers, GH3-10f and GH3-10r, shown below, which weredesigned based on the publicly available Aspergillus clavatus partialmRNA sequence (XM_001269581) for direct cloning by IN-FUSION™ strategy.

Primer GH3-10f: (SEQ ID NO: 231)acacaactggggatccaccATGAGGTTCAGCTGGCTTGAGGTCG Primer GH3-10r:(SEQ ID NO: 232) agatctcgagaagcttaCTGTACCCGGGGCAGAGGTGCTCTC

A PCR reaction was performed with the genomic DNA prepared fromAspergillus clavatus NRRL1 in order to amplify the full-length gene. ThePCR reaction was composed of 1 μl of genomic DNA, 0.75 μl of primerGH3-10f (10 μM), 0.75 μl of primer GH3-10r (10 μM), 3 μl of 5×HF buffer,0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μl of PHUSION® DNApolymerase, and PCR-grade water up to 15 μl. The PCR reaction wasperformed using a DYAD® PCR machine programmed for 2 minutes at 98° C.followed by 10 touchdown cycles at 98° C. for 15 seconds, 70° C. (−1°C./cycle) for 30 seconds, and 72° C. for 2 minutes 30 seconds; and 25cycles each at 98° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for2 minutes 30 seconds, and 5 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 3.0 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Aspergillus clavatus beta-glucosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an I N-FUSION™ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Aspergillus clavatusbeta-glucosidase gene sequence was verified by Sanger sequencing beforeheterologous expression.

The coding sequence is 3062 bp including the stop codon and isinterrupted by 8 introns of 67 bp (nucleotides 58 to 124), 61 bp(nucleotides 265 to 325), 62 bp (nucleotides 371 to 432), 65 bp(nucleotides 489 to 553), 50 bp (nucleotides 948 to 997), 53 bp(nucleotides 1021 to 1073), 61 bp (nucleotides 1849 to 1909), and 60 bp(nucleotides 2769 to 2828). The encoded predicted protein is 860 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 18 residues was predicted. Thepredicted mature protein contains 842 amino acids.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 were added and gently mixed. The mixture was incubated at 37° C. for30 minutes and the protoplasts were spread on COVE sucrose (1 M) platessupplemented with 10 mM acetamide and 15 mM CsCl for transformantselection. After incubation for 4-7 days at 37° C. spores of severaltransformants were seeded on YP-2% maltodextrin medium. After 4 dayscultivation at 30° C. culture broth was analyzed in order to identifythe best transformants based on their ability to produce a large amountof active Aspergillus clavatus beta-glycosidase. The screening was basedon intensity of the band corresponding to the heterologous expressedprotein determined by SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-glucopyranoside (pNPG) as described in Example 16.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then carried out in 250 ml shake flasks using YP-2% maltodextrinmedium for 4 days at 30° C. with shaking at 100 rpm. The broth wasfiltered using standard methods. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 76: Preparation of Thielavia terrestris NRRL 8126 GH3Beta-Glucosidase

The Thielavia terrestris GH3 beta-glucosidase (SEQ ID NO: 183 [DNAsequence] and SEQ ID NO: 184 [deduced amino acid sequence]) was obtainedaccording to the procedure described below.

Three agarose plugs from culture of Thielavia terrestris NRRL 8126 grownon a PDA plate were inoculated into 100 ml of NNCYP medium supplementedwith 1.5% glucose and incubated for 25 hours at 42° C. and 200 rpm on anorbital shaker. Fifty ml of this culture was used to inoculate 1.8 literof NNCYP medium supplemented with 0.4% glucose and 52 g of powderedcellulose per liter and was incubated at 42° C. The pH was controlled at5.0 by the addition of 15% ammonium hydroxide or 5 N phosphoric acid, asneeded.

The fermentations were run at 42° C. with minimum dissolved oxygen at25% at a 1.0 VVM air flow and an agitation at 1100 rpm. Feed medium wasdelivered into a 2 liter fermentation vessel at 0 hours with a feed rateof 6.0-8.0 g/hour for 120 hours. Pooled cultures were centrifuged at3000×g for 10 minutes and the supernatant was filtered through adisposable filtering unit with a glass fiber prefilter (Nalgene,Rochester N.Y., USA). The filtrate was cooled to 4° C. for storage.

A 0.3 ml aliquot of the filtrate was precipitated with 10%trichloroacetic acid (TCA)-80% acetone for 20 minutes on ice. Thesuspension was centrifuged for 10 minutes at 13,000×g. The supernatantwas removed and the protein pellet remaining was rinsed with coldacetone. The protein pellet was dissolved in 30 μl of 1× lithium dodecylsulfate (LDS) SDS-PAGE loading buffer with 50 mM dithiothreitol (DTT)and heated at 80° C. for 10 minutes. A 15 μl sample was separated bySDS-PAGE using a 7 cm 4-12% NuPAGE Bis-Tris SDS-PAGE gradient gel and2-(N-morpholino)ethanesulfonic acid (MES) running buffer. The SDS-PAGEwas run under reducing conditions according to the manufacturer'srecommended protocol (Invitrogen, Carlsbad, Calif., USA). The gel wasremoved from the cassette and rinsed 3 times with deionized water for atleast 5 minutes each and stained with Bio-Safe Coomassie Stain (Bio-RadLaboratories, Inc., Hercules, Calif., USA) for 1 hour followed bydestaining with doubly-distilled water for more than 30 minutes. Proteinbands observed at approximately 95 kD was excised and reduced with 50 μlof 10 mM DTT in 100 mM ammonium bicarbonate for 30 minutes. Followingreduction, the gel pieces were alkylated with 50 μl of 55 mMiodoacetamide in 100 mM ammonium bicarbonate for 20 minutes. The driedgel pieces were allowed to re-hydrate in a trypsin digestion solution (6ng/μ1 sequencing grade trypsin in 50 mM ammonium bicarbonate) for 30minutes at room temperature, followed by an 8 hour digestion at 40° C.Each of the reaction steps described was followed by numerous washes andpre-washes with the desired solutions. Fifty μl of acetonitrile was usedto de-hydrate the gel pieces between reactions and they were air-driedbetween steps. Peptides were extracted twice with 1% formic acid/2%acetonitrile in HPLC grade water for 30 minutes. Peptide extractionsolutions were transferred to a 96 well PCR type microtiter plate thathad been cooled to 10-15° C. Microtiter plates containing the recoveredpeptide solutions were sealed to prevent evaporation and stored at 4° C.until mass spectrometry analysis could be performed.

For de-novo peptide sequencing by tandem mass spectrometry, a Q-Tofmicron“, a hybrid orthogonal quadrupole time-of-flight mass spectrometer(Waters Micromass® MS Technologies, Milford, Mass., USA) was used forLC/MS/MS analysis. The Q-Tof micron” was fitted with an Ultimate™capillary and nano-flow HPLC system which had been coupled with a FAMOSmicro autosampler and a Switchos II column switching device (LCPackings,San Francisco, Calif., USA) for concentrating and desalting samples.Samples were loaded onto a guard column (300 μm ID×5 cm, C18 pepmap)fitted in the injection loop and washed with 0.1% formic acid in waterat 40 μl/minute for 2 minutes using the Switchos II pump. Peptides wereseparated on a 75 μm ID×15 cm, C18, 3 μm, 100 Å PepMap™ (LC Packings,San Francisco, Calif., USA) nanoflow fused capillary column at a flowrate of 175 nI/minute from a split flow of 175 μl/minute using a NAN-75calibrator (Dionex, Sunnyvale, Calif., USA). A step elution gradient of5% to 80% acetonitrile in 0.1% formic acid was applied over a 45 minuteinterval. The column eluent was monitored at 215 nm and introduced intothe Q-Tof Micron™ through an electrospray ion source fitted with thenanospray interface. The Q-Tof Micron™ is fully microprocessorcontrolled using Masslynx™ software version 3.5 (Waters Micromass® MSTechnologies, Milford, Mass., USA). Data was acquired in survey scanmode and from a mass range of m/z 400 to 1990 with the switchingcriteria for MS to MS/MS to include an ion intensity of greater than10.0 counts per second and charge states of +2, +3, and +4. Analysisspectra of up to 4 co-eluting species with a scan time of 1.9 secondsand inter-scan time of 0.1 seconds could be obtained. A cone voltage of65 volts was typically used and the collision energy was programmed tobe varied according to the mass and charge state of the eluting peptideand in the range of 10-60 volts. The acquired spectra were combined,smoothed and centered in an automated fashion and a peak list generated.This peak list was searched against selected public and privatedatabases using ProteinLynx™ Global Server 1.1 software (WatersMicromass® MS Technologies, Milford, Mass.). Results from theProteinLynx™ searches were evaluated and un-identified proteins wereanalyzed further by evaluating the MS/MS spectrums of each ion ofinterest and de-novo sequence was determined by identifying the y and bion series and matching mass differences to the appropriate amino acid.

Peptide sequences obtained from de novo sequencing by mass spectrometrywere obtained from several multiply charged ions for the approximately95 kDa polypeptide gel band.

A doubly charged tryptic peptide ion of 524.76 m/z sequence wasdetermined to be Ser-Pro-Phe-Thr-Trp-Gly-Pro-Thr-Arg (amino acids 607 to615 of SEQ ID NO: 184). A second doubly charged tryptic peptide ion of709.91 partial sequence was determined to beGly-Val-Asn-Val-[Ile/Leu]-[Ile-Leu]-Gly-[Ile/Leu]-Gly-Pro (amino acids148 to 155 of SEQ ID NO: 184). A second doubly charged tryptic peptideion of 745.38 partial sequence was determined to bePro-Pro-His-Ala-Thr-Asp (amino acids 747 to 752 of SEQ ID NO: 184). Athird doubly charged tryptic peptide ion of 808.92 m/z sequence wasdetermined to beTyr-Glu-Ser-[Ile/Leu]-[Ile/Leu]-Ser-Asn-Tyr-Ala-Thr-Ser-Qln-[Ile/Leu]-Lys(amino acids 487 to 499 of SEQ ID NO: 184). A fourth doubly chargedtryptic peptide ion of 1023.96 a partial sequence was determined to bePhe-Asn-Ser-Gly-Phe-Pro-Ser-Gly-Gln-Thr-Ala-Ala-Ala-Thr-Phe-Asp-Arg(amino acids 114 to 130 of SEQ ID NO: 184).

To generate genomic DNA for PCR amplification, Thielavia terrestris NRRL8126 was grown in 50 ml of NNCYP medium supplemented with 1% glucose ina baffled shake flask at 42° C. and 200 rpm for 24 hours. Mycelia wereharvested by filtration, washed twice in TE (10 mM Tris-1 mM EDTA), andfrozen under liquid nitrogen. A pea-size piece of frozen mycelia wassuspended in 0.7 ml of 1% lithium dodecyl sulfate in TE and disrupted byagitation with an equal volume of 0.1 mm zirconia/silica beads (BiospecProducts, Inc., Bartlesville, Okla., USA) for 45 seconds in a FastPrepFP120 (ThermoSavant, Holbrook, N.Y., USA). Debris was removed bycentrifugation at 13,000×g for 10 minutes and the cleared supernatantwas brought to 2.5 M ammonium acetate and incubated on ice for 20minutes. After the incubation period, the nucleic acids wereprecipitated by addition of 2 volumes of ethanol. After centrifugationfor 15 minutes in a microfuge at 4° C., the pellet was washed in 70%ethanol and air dried. The DNA was resuspended in 120 μl of 0.1×TE andincubated with 1 μl of DNase-free RNase A at 37° C. for 20 minutes.Ammonium acetate was added to 2.5 M and the DNA was precipitated with 2volumes of ethanol. The pellet was washed in 70% ethanol, air dried, andresuspended in TE buffer.

A low redundancy draft sequence of the Thielavia terrestris NRRL 8126genome was generated by the Joint Genome Center (JGI), Walnut Creek,Calif., USA, using the whole genome shotgun method according to Martinezet al., 2008, Nature Biotechnol. 26: 553-560. Shotgun sequencing reads(approximately 18307) were assembled into contigs using the Phrapassembler (Ewing and Green, 1998, Genome Res. 8: 186-194).

A tblastn search (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402) of the assembled contigs was carried out using as query abeta-glucosidase protein sequence from Neurospora crassa (UniProtaccession number q7rwp2). A translated amino acid sequence with greaterthan 73.8% identity to the query sequence was identified.

The sequence was searched against public databases using blastp(Altschul et al., 1997, supra) and was found to be 80.1% identical to abeta-glucosidase from Podospora anserina (UniProt accession numberB2AVE8).

The nucleotide sequence and deduced amino acid sequence of the Thielaviaterrestris beta-glucosidase gene are shown in SEQ ID NO: 183 and SEQ IDNO: 184, respectively. The coding sequence is 3032 bp including the stopcodon and is interrupted by three introns of 295 bp, 57 bp, and 61 bp.The encoded predicted protein is 872 amino acids. Using the SignalPprogram (Nielsen et al., 1997, Protein Engineering 10: 1-6), a signalpeptide of 18 residues was predicted. The predicted mature proteincontains 854 amino acids with a predicted molecular mass of 93 kDa andan isoelectric point of 5.57.

Thielavia terrestris NRRL 8126 was grown for 3 days on PDA plates at 45°C. Mycelia were scraped from the plates and approximately 100 mg ofmycelia (wet weight) were used to inoculate 500 ml shake flaskscontaining 100 ml of either MY50 medium or 1× Vogel's mediumsupplemented with 2% microcrystalline cellulose (AVICEL®). Cultures weregrown for 2, 3, 4 and 5 days at 45° C. with vigorous shaking. Themycelia were harvested by filtration through MIRACLOTH® (EMD ChemicalsInc., Gibbstown, N.J., USA), and quick frozen in liquid nitrogen.

To isolate total RNA frozen mycelia of Thielavia terrestris NRRL 8126were ground in an electric coffee grinder. The ground material was mixed1:1 v/v with 20 ml of FENAZOL™ (Ambion, Inc., Austin, Tex., USA) in a 50ml tube. Once the mycelia were suspended, they were extracted withchloroform and three times with a mixture of phenol-chloroform-isoamylalcohol 25:24:1 v/v/v. From the resulting aqueous phase, the RNA wasprecipitated by adding 1/10 volume of 3 M sodium acetate pH 5.2 and 1.25volumes of isopropanol. The precipitated RNA was recovered bycentrifugation at 12,000×g for 30 minutes at 4° C. The final pellet waswashed with cold 70% ethanol, air dried, and resuspended in 500 ml ofdiethylpyrocarbonate treated water (DEPC-water).

RNA samples were pooled and the quality and quantity of the purified RNAwas assessed with an AGILENT® 2100 Bioanalyzer (Agilent Technologies,Inc., Palo Alto, Calif., USA). A total of 630 μg of RNA was isolated.

Poly A+RNA was isolated from total RNA using an Absolutely mRNA™Purification Kit (Stratagene, La Jolla, Calif., USA) according to themanufacturer's instructions. cDNA synthesis and cloning was performedaccording to a procedure based on the SuperScript™ Plasmid System withGateway® Technology for cDNA Synthesis and Cloning (Invitrogen, Carsbad,Calif., USA). 1-2 μg of poly A+RNA, reverse transcriptase SuperScript II(Invitrogen, Carsbad, Calif., USA), and oligo dT-Not I primer5′-GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTVN-3′ (SEQ ID NO: 233)were used to synthesize first strand cDNA. Second strand synthesis wasperformed with E. coli DNA ligase, polymerase I, and RNase H followed byend repair using T4 DNA polymerase. The Sal I adaptor(5′-TCGACCCACGCGTCCG-3′ [SEQ ID NO: 234] and 5′-CGGACGCGTGGG-3′ [SEQ IDNO: 235]) was ligated to the cDNA, digested with Not I, and subsequentlysize selected (>2 kb) by 1.1% agarose gel electrophoresis using TAEbuffer. The cDNA inserts were directionally ligated into Sal I and Not Idigested vector pCMVsport6 (Invitrogen, Carsbad, Calif., USA). Theligation was transformed into ElectroMAX™ DH10B™ T1 cells (Invitrogen,Carsbad, Calif., USA).

Library quality was first assessed by randomly selecting 24 clones andPCR amplifying the cDNA inserts with the primers M13-F and M13-R shownbelow to determine the fraction of insertless clones.

Primer M13-F: (SEQ iD NO: 236) 5′-GTAAAACGACGGCCAGT-3′ Primer M13-R:(SEQ iD NO: 237) 5′-AGGAAACAGCTATGACCAT-3′

Colonies from each library were plated onto LB plates at a density ofapproximately 1000 colonies per plate. Plates were grown at 37° C. for18 hours and then individual colonies were picked and each used toinoculate a well containing LB medium supplemented with 100 μg ofampicillin per ml in a 384 well plate (Nunc, Rochester, N.Y., USA).Clones were grown at 37° C. for 18 hours. Plasmid DNA for sequencing wasproduced by rolling circle amplification using a Templiphi™ Kit (GEHealthcare, Piscataway, N.J., USA). Subclone inserts were sequenced fromboth ends using primers complimentary to the flanking vector sequence asshown below and BigDye® terminator chemistry using a 3730 DNA Analyzer(Applied Biosystems, Foster City, Calif., USA).

Forward primer: (SEQ iD NO: 238) 5′-ATTTAGGTGACACTATAGAA-3′Reverse primer: (SEQ ID NO: 239) 5′-TAATACGACTCACTATAGGG-3′

A clone showing 67.4% identity at the nucleotide level to abeta-glucosidase from Aspergillus oryzae (U.S. Published Application No.2005233423) was identified.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Thielavia terrestris Family GH3B beta-glucosidase gene fromthe cDNA clone. An IN-FUSION® Cloning Kit (BD Biosciences, Palo Alto,Calif., USA) was used to clone the fragment directly into the expressionvector pAILo2 (WO 2005/074647), without the need for restrictiondigestion and ligation.

Forward primer: (SEQ ID NO: 240) 5′-ACTGGATTTACCATGAAGCCTGCCATTGTGCT-3′Reverse primer: (SEQ ID NO: 241)5′-TCACCTCTAGTTAATTAATCACGGCAACTCAATGCTCA-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 200 ng of plasmid cDNA, 1× 2× Advantage GC-Melt LA Buffer(Clonetech Laboratories, Inc., Mountain View, Calif., USA), 1 μl of 10mM blend of dATP, dTTP, dGTP, and dCTP, and 1.25 units of Advantage GCGenomic LA Polymerase Mix (Clonetech Laboratories, Inc., Mountain View,Calif., USA) in a final volume of 50 μl. The amplification was performedusing an EPPENDORF® MASTERCYCLER® 5333 epgradient S (EppendorfScientific, Inc., Westbury, N.Y., USA) programmed for one cycle at 94°C. for 1 minute; and 30 cycles each at 94° C. for 30 seconds, 60.5° C.for 30 seconds, and 72° C. for 3 minutes. The heat block was then heldat 72° C. for 15 minutes followed by a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.6 kb product band was excisedfrom the gel and purified using a MINELUTE® Gel Extraction Kit (QIAGENInc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The fragment was then cloned into pAILo2 using an IN-FUSION® CloningKit. The vector was digested with Nco I and Pac I. The fragment waspurified by gel electrophoresis as above and a QIAQUICK® GelPurification Kit (QIAGEN Inc., Valencia, Calif., USA). The gene fragmentand the digested vector were combined together in a reaction resultingin the expression plasmid pAG81, in which transcription of the FamilyGH3B protein gene was under the control of the NA2-tpi promoter (ahybrid of the promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus nidulans triose phosphate isomerase). Therecombination reaction (10 μl) was composed of 1× IN-FUSION® Buffer (BDBiosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto,Calif., USA), 1 μl of IN-FUSION® enzyme (diluted 1:10) (BD Biosciences,Palo Alto, Calif., USA), 108 ng of pAILo2 digested with Nco I and Pac 1,and 94 ng of the Thielavia terrestris GH3B protein purified PCR product.The reaction was incubated at 37° C. for 15 minutes followed by 15minutes at 50° C. The reaction was diluted with 40 μl of 10 mM Tris-0.1M EDTA buffer and 2.5 μl of the diluted reaction was used to transformE. coli TOP10 Competent cells. An E. coli transformant containing pAG81(GH3B protein gene) was identified by restriction enzyme digestion andplasmid DNA was prepared using a BIOROBOT® 9600. The plasmid constructwas sequenced using an Applied Biosystems 3130xl Genetic Analyzer(Applied Biosystems, Foster City, Calif., USA) to verify the genesequence.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422, whichwere transformed with 5 μg of pAG81. Twenty-six transformants wereisolated to individual PDA plates. A small plug from each transformantwas used to inoculate 1 ml of M410 media in a 24 well plate andincubated at 34° C. After 5 days of incubation, 7.5 μl of supernatantfrom four transformants was analyzed using a CRITERION® stain-free,8-16% gradient SDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that several transformants had a newmajor band of approximately 150 kDa.

A confluent PDA plate of the top transformant (was washed with 5 ml of0.01% TWEEN® 20 and inoculated into 500 ml flasks each containing 100 mlof M410 medium to generate broth for characterization of the enzyme. Theflasks were harvested on day 5, filtered using a 0.22 μm stericupsuction filter (Millipore, Bedford, Mass., USA), and stored at 4° C.

A 45 ml aliquot of shake flask broth prepared was concentrated ten-foldusing a VIVASPIN™ centrifugal concentrator with a molecular weightcut-off of 10 kDa and buffer-exchanged into 50 mM sodium acetate of pH5. A 4 ml aliquot of the buffer-exchanged, concentrated broth was thendiluted to a 10 ml volume with 1 M Tris-HCl pH 8 and water to aconcentration of 20 mM Tris-HCl, and the pH adjusted to a final value of8 using 2 N sodium hydroxide. The resulting material was then purifiedusing a MONO Q™ 5/5 column (GE Healthcare, Piscataway, N.J., USA)equilibrated with 20 mM Tris-HCl buffer pH 7.8. Unbound material waswashed from the column with 2 ml equilibration buffer, and then thecolumn was eluted with a linear gradient from 0-500 mM sodium chloridein the equilibration buffer. Fractions of 0.3 ml were collected. Unboundmaterial collected during column loading and unbound material washedfrom the column were pooled for a total volume of 12 ml. Five μl of thefractions, including the pooled unbound material, showing UV absorbanceat 280 nm were analyzed using a CRITERION STAIN FREE™ 8-16% Tris-HClSDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules, Calif., USA)according to the manufacturer's instructions. Precision Plus Protein™unstained standards (Bio-Rad Laboratories, Inc., Hercules, Calif., USA)were used as molecular weight markers. The gel was removed from thecassette and imaged using a CRITERION STAIN FREE™ IMAGER (Bio-RadLaboratories, Inc., Hercules, Calif., USA). The beta-glucosidase wasidentified in the pooled unbound material as a band at 150 kDa bySDS-PAGE. The 12 ml of pooled unbound material was concentrated asdescribed above to a final volume of 2 ml. The concentrated material waspurified using a HI LOAD™ 16/60 SUPERDEX™ 75 PREP GRADE column (GEHealthcare, Piscataway, N.J., USA) equilibrated with 20 mM Tris.HClbuffer of pH 7.8 containing 150 mM sodium chloride, and eluted with theequilibration buffer. Fractions of 3 ml were collected. Column fractionswere analyzed by SDS-PAGE as described above. Fractions containing thepurified beta-glucosidase, identified by a band at 150 kDa by SDS-PAGE,were pooled for a total volume of 6 ml. The pooled material wasconcentrated using a VIVASPIN™ centrifugal concentrator with a molecularweight cut-off of 5 kDa (GE Healthcare, Piscataway, N.J., USA) to afinal volume of 0.4 ml. Protein concentration was determined using theBio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA).

The enzyme activity of the purified beta-glucosidase was measured usingp-nitrophenyl-beta-D-glucopyranoside as substrate. Ap-nitrophenyl-beta-D-glucopyranoside stock solution was made bydissolving p-nitrophenyl-beta-D-glucopyranoside in dimethylsulfoxide(DMSO) to constitute a 0.1 M solution. Before assay, a sample of thestock solution was diluted 100-fold in 100 mM sodium acetate pH 5containing 0.01% TWEEN® 20 to a 1 mM solution. A 100 μl volume of 1 mMp-nitrophenyl-beta-D-glucopyranoside was mixed with each dilution of theenzyme for a 120 μl total volume, and then incubated at 40° C. for 20minutes. Substrate alone, enzyme alone, and buffer alone were run ascontrols. p-Nitrophenol standard solutions of 0.40, 0.25, 0.20, 0.10,0.05, and 0.02 mM were prepared by diluting a 10 mM stock solution in100 mM sodium acetate pH 5 containing 0.01% TWEEN® 20. At 20 minutes, 50μl of 1.0 M sodium carbonate buffer pH 10 was added to each well(including samples, substrate control, enzyme control, reagent control,and standards), mixed, and the absorbance at 405 nm immediately measuredon a SPECTRAMAX™ 340 PC plate reader (Molecular Devices, Sunnyvale,Calif., USA). The activity measured was 84 units per mg of protein. Oneunit of activity was defined as the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 40° C.

The enzyme activity of the purified beta-glucosidase was also measuredwith cellobiose. A 2 mg/ml stock solution of cellobiose was prepared in50 mM sodium acetate pH 5 containing 0.01% TWEEN® 20, and 100 μl werecombined with each enzyme dilution for a total volume of 150 μl andincubated at 50° C. for 30 minutes. Substrate alone, enzyme alone, andbuffer alone were run as controls. Glucose standard solutions of 1.0,0.50, 0.25, 0.125, 0.063, and 0.031 mg/ml were prepared in 50 mM sodiumacetate pH 5. containing 0.01% TWEEN® 20. At 30 minutes, 50 μl of 0.5 Msodium hydroxide solution was added to each well (including samples,substrate control, enzyme control, reagent control, and standards),mixed, and then 20 μl of the mixture was transferred to a Costar®EIA/RIA 96-well plate (Corning Incorporated, Corning, N.Y., USA) andcombined with 200 μl of GLUCOSE OXIDASE REAGENT (Pointe Scientific,Inc., Canton, Mich., USA), and allowed to stand at 25° C. for 20 minutesand then the absorbance at 500 nm was measured on a SPECTRAMAX™ 340 PCplate reader (Molecular Devices, Sunnyvale, Calif., USA). The activitymeasured was 264 units per mg of protein. One unit of activity wasdefined as the amount of enzyme capable of releasing 1 μmole of glucoseper minute at pH 5, 50° C.

Example 77: Preparation of Penicillium oxalicum Strain IBT5387 GH3Beta-Glucosidase

The Penicillium oxalicum strain IBT5387 (Technical University ofDenmark; NN005786) GH3 beta-glucosidase (SEQ ID NO: 185 [DNA sequence]and SEQ ID NO: 186 [deduced amino acid sequence]) was obtained accordingto the procedure described below.

Aspergillus oryzae BECh2 (WO 2000/39322) was used as a host cell forexpressing the P. oxalicum strain IBT5387 Family GH3 beta-glucosidasegene.

A set of degenerate primers shown below were designed according to thestrategy described by Rose et al., 1998, Nucleic Acids Research 26:1628-1635, for cloning a gene encoding a beta-glucosidase (EC 3.2.1.21)belonging to Family GH3.

GH3scree.f1: (SEQ ID NO: 242) atgaccctggccgaaaaagtcaacytnacnacnggGH3scree.f2: (SEQ ID NO: 243) ggtggccggaactgggaaggcttctsnccngayccGH3scree.f5: (SEQ ID NO: 244) gagctgggcttccagggctttgtnatgwsngaytggGH3scree.f6: (SEQ ID NO: 245) agcgctttggccggcctcgayatgwsnatgccGH3scree.r1: (SEQ ID NO: 246) atcccagttgctcaggtcccknckngt GH3scree.r2:(SEQ ID NO: 247) aaaggttgtgtagctcagnccrtgnccraaytc GH3scree.r3:(SEQ ID NO: 248) gtcaaagtggcggtagtcgatraanacnccytc GH3scree.r4:(SEQ ID NO: 249) ggtgggcgagttgccgacggggttgactctgccrtanar GH3scree.r5:(SEQ ID NO: 250) gccgggcagaccggcccagaggatggcngtnacrttngg GH3scree.r6:(SEQ ID NO: 251) caggacggggccaaccgagtgaatgacnacdatngtrtt

PCR screening of Penicillium oxalicum strain IBT5387 was performed usingtwo successive PCRs. The forward primers (GH3scree.f1, GH3scree.f2,GH3scree.f5, or GH3scree.f6) (0.33 μl of a 10 mM stock) were combinedwith the reverse primers (GH3scree.r1, GH3scree.r2, GH3scree.r3,GH3scree.r4, GH3scree.r5, or GH3scree.r6) (0.33 μl of a 10 mM stock) ina 10 μl mixture containing 0.33 μl of P. oxalicum genomic DNA and 5 μlof REDDYMIX™ Extensor PCR Master Mix 1 (ABgene Ltd., Surrey, UnitedKingdom). P. oxalicum genomic DNA was obtained according to theprocedure described in the FastDNA® SPIN Kit (Q-BIOgene, Carlsbad,Calif., USA). The PCR reaction was performed using a DYAD® ThermalCycler (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) programmedfor one cycle at 94° C. for 2 minutes; 9 cycles each at 94° C. for 15seconds, 63° C. for 30 seconds with a decrease of 1° C. for each cycle,and 68° C. for 1 minutes 45 seconds; 24 cycles each at 94° C. for 15seconds, 55° C. for 30 seconds, and 68° C. for 1 minutes 45 seconds; andextension at 68° C. for 7 minutes.

PCR products obtained during the first PCR were re-amplified with theircorresponding primers by transferring 0.5 μl of the first PCR reactionto a second 20 μl mixture containing the same concentration of primers,dNTPs, DNA polymerase, and buffer as the first PCR reaction. The secondPCR was performed using a DYAD® Thermal Cycler programmed for one cycleat 94° C. for 2 minutes; 24 cycles each at 94° C. for 15 seconds, 58° C.for 30 seconds, and 68° C. for 1 minutes 45 seconds; and an extension at68° C. for 7 minutes.

PCR products obtained during the second amplification were analyzed by1% agarose gel electrophoresis using TAE buffer. Single bands rangingfrom 2000 to 800 nucleotides in size were excised from the gel andpurified using a GFX® PCR DNA and Gel Band Purification Kit according tothe manufacturer's instructions. Purified DNA samples were directlysequenced with primers used for amplification. Sequences were assembledin SeqMan v7.2.1 (DNA star, Madison, Wis., USA) into a contig that wasused for designing primers shown below, based on recommendations oflength and temperature described in a GENE WALKING SPEEDUP™ Kit(Seegene, Inc., Seoul, Korea).

5786GH3-51TSP1f: (SEQ ID NO: 252) CACCAACACCGGCAATCTAGC 5786GH3-51TSP2f:(SEQ ID NO: 253) GGTGACGAGGTTGTCCAACTGTACG 5786GH3-51TSP1r:(SEQ ID NO: 254) CTTGAAGCCAAGGCGAGG 5786GH3-51TSP2r: (SEQ ID NO: 255)TCCGGTATTTCCTACACATGGTCC

GeneWalking was based on the protocol from the GENE WALKING SPEEDUP™ Kitwith some minor differences. Only two PCR amplifications were carriedout and in both cases, the REDDYMIX™ Extensor PCR Master Mix 1 was usedin place of the enzyme mix present in the Kit. GeneWalking PCR 1 wasperformed in a total volume of 15 μl by mixing 1.2 μl of primer 1 to 4(2.5 mM) from the GENE WALKING SPEEDUP™ Kit with 0.3 μl of primer5786GH3-51TSP1f or primer 5786GH3-51TSP1r (10 mM) in the presence of 7.5μl of REDDYMIX™ Extensor PCR Master Mix 1, and 0.5 μl of P. oxalicumgenomic DNA. The PCR was performed using a DYAD® Thermal Cyclerprogrammed for one cycle at 94° C. for 3 minutes followed by 1 minute at42° C. and 2 minutes at 68° C.; 30 cycles each at 94° C. for 30 seconds,58° C. for 30 seconds, and 68° C. for 1 minute and 40 seconds; andelongation at 68° C. for 7 minutes. A 0.5 μl aliquot of theamplification reaction was transferred to a second PCR tube containing a20 μl mixture composed of 10 μl of REDDYMIX™ Extensor PCR Master Mix 1,1 μl of primer 5 (10 mM) from the Kit, 1 μl of primers T5786GH3-51SP2for 5786GH3-51TSP2r (10 mM). The amplification was performed in a DYAD®Thermal Cycler programmed for denaturation at 94° C. for 3 minutes; 35cycles each at 94° C. for 30 seconds, 58° C. for 30 seconds, and 68° C.for 1 minute and 40 seconds; and elongation at 68° C. for 7 minutes.

The PCR products were analyzed by 1% agarose gel electrophoresis in TAEbuffer. Single bands ranging from 700 to 1200 nucleotides in size wereexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. Purified DNAsamples were directly sequenced with the primers used for amplification.

Based on blastx analyses, the start and the stop codons of the gene wereidentified and the primers shown below were designed for cloning thegene into the expression vector pDAu109 (WO 2005042735) using anIN-FUSION™ Dry-Down PCR Cloning Kit.

5786GH3-51r1: (SEQ ID NO: 256)agatctcgagaagcttaCCATGACTCCAATCGCGCGCTCAAGG 5786GH3-51f1:(SEQ ID NO: 257) acacaactggggatccaccATGAGGAGCTCAACGACGGTTCTGGCC

The P. oxalicum beta-glucosidase gene was amplified by PCR using the twocloning primers described above with P. oxalicum strain IBT5387 genomicDNA. The PCR was composed of 1 μl of P. oxalicum genomic DNA, 0.75 μl ofprimer 5786GH3-51f1 (10 μM), 0.75 μl of primer 5786GH3-51r1 (10 μM), 3μl of 5×HF buffer, 0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μlof PHUSION® DNA polymerase, and PCR-grade water to 15 μl. Theamplification reaction was performed using a DYAD® Thermal Cyclerprogrammed for 2 minutes at 98° C. followed by 10 touchdown cycles eachat 98° C. for 15 seconds, 70° C. (−1° C./cycle) for 30 seconds, and 72°C. for 2 minutes and 30 seconds; and 25 cycles each at 98° C. for 15seconds, 60° C. for 30 seconds, 72° C. for 2 minutes and 30 seconds, and5 minutes at 72° C.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.8 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Penicillium oxalicum beta-glucosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an IN-FUSION™′ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions. A 2.5 μl volume of thediluted ligation mixture was used to transform E. coli TOP10 chemicallycompetent cells. Three colonies were selected on LB agar platescontaining 100 μg of ampicillin per ml and cultivated overnight in 3 mlof LB medium supplemented with 100 μg of ampicillin per ml. Plasmid DNAwas purified using an E.Z.N.A.® Plasmid Mini Kit according to themanufacturer's instructions. The Penicillium oxalicum beta-glucosidasegene sequence was verified by Sanger sequencing before heterologousexpression. One plasmid designated pIF113 #1 was selected for expressingthe P. oxalicum beta-glucosidase in an Aspergillus oryzae host cell.

The coding sequence is 2793 bp including the stop codon with 2 predictedintrons of 82 bp (nucleotides 85 to 116) and 59 bp (nucleotides 346 to404). The encoded predicted protein is 883 amino acids. Using theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6), asignal peptide of 21 residues was predicted. The predicted matureprotein contains 862 amino acids with a predicted molecular mass of 94.7kDa and an isoelectric point of 5.04.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were preparedaccording to WO 95/002043. One hundred μl of protoplasts were mixed with2.5-15 μg of the Aspergillus expression vector and 250 μl of 60% PEG4000 (Applichem, Darmstadt, Germany) (polyethylene glycol, molecularweight 4,000), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 and gently mixed.The mixture was incubated at 37° C. for 30 minutes and the protoplastswere spread onto COVE plates for selection. After incubation for 4-7days at 37° C. spores of sixteen transformants were inoculated into 0.5ml of YP medium supplemented with 2% maltodextrin in 96 deep-wellplates. After 4 days cultivation at 30° C., the culture broths wereanalyzed to identify the best transformants producing large amounts ofactive P. oxalicum beta-glucosidase. The analysis was based on SDS-PAGEand activity of the enzyme on 4-nitrophenyl-beta-D-glucopyranoside(pNPG) as described in Example 16.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE plates containing 10 mM sodiumnitrate. Spores were then inoculated into 100 ml of YP mediumsupplemented with 2% maltodextrin in 250 ml shake flasks and incubatedfor 4 days at 30° C. with shaking at 100 rpm.

Combined supernatants of the shake flask cultures were first filteredusing a glass micro fiber filter with a 0.7 μm pore size, and thensterile filtered using a filtration unit equipped with a PES (Polyethersulfone) with a 0.22 μm pore size (Nalge Nunc International, New York,N.Y. USA). The sterile filtered supernatant was then adjusted to aconcentration of 2 M ammonium sulfate by slowly adding solid ammoniumsulfate, dissolving by gentle stirring, and then filtering using a glassmicro fiber filter with a 0.7 μm pore size. If there was anyprecipitate, it was discarded.

The filtered supernatant was applied to a 50 ml Toyopearl Phenyl-650Mcolumn (TOSOH Bioscience GmbH, Germany) equilibrated with 2 M ammoniumsulfate in water and unbound material was eluted with 2 M ammoniumsulfate until the UV absorbance at 280 nm was below 0.05. Bound proteinwas eluted with 50% ethanol as solution B using a step gradient. Ten mlfractions were collected and moniterd by UV absorbance at 280 nm. Thefractions were analyzed by SDS-PAGE and pooled the fractions containingprotein with the expected molecular weight. The pooled fractions weredialyzed using 50 mM HEPES pH 7.5 buffer, so the ionic strength wasbelow 4 MSi and the pH was 7.5.

The pooled protein was applied to a 50 ml Q Sepharose® Fast Flow columnequilibrated with 50 mM HEPES pH 7.5 buffer and unbound material waseluted with 50 mM HEPES buffer pH 7.5. The bound protein was then elutedwith a linear 20 column volume salt gradient in 50 mM HEPES buffer pH7.5 containing 1 M NaCl as buffer B. Ten ml fractions of the eluate werecollected and each were analyzed for purity of the protein by SDS-PAGE.The fractions with highest purity were pooled. Identity of thebeta-glucosidase was confirmed by mass spectroscopy and proteinidentification was carried out by in gel digestion. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 78: Preparation of Penicillium oxalicum Strain IBT5387 GH3Beta-Glucosidase

The Penicillium oxalicum strain IBT5387 (Technical University ofDenmark; NN005786) GH3 beta-glucosidase (SEQ ID NO: 187 [DNA sequence]and SEQ ID NO: 188 [deduced amino acid sequence]) was obtained accordingto the procedure described below.

Genomic DNA was isolated according to the procedure described in Example73.

A set of degenerate primers (shown below) was hand-designed according tothe strategy described by Rose et al., 1998, Nucleic Acids Res. 26:1628-1635, for targetting beta-glucosidases (EC 3.2.1.21) belonging toFamily GH3.

GH3scree.f1 (SEQ ID NO: 258) atgaccctggccgaaaaagtcaacytnacnacnggGH3scree.f2 (SEQ ID NO: 259) ggtggccggaactgggaaggcttctsnccngayccGH3scree.f5 (SEQ ID NO: 260) gagctgggcttccagggctttgtnatgwsngaytggGH3scree.f6 (SEQ ID NO: 261) agcgctttggccggcctcgayatgwsnatgccGH3scree.r1 (SEQ ID NO: 262) atcccagttgctcaggtcccknckngt GH3scree.r2(SEQ ID NO: 263) aaaggttgtgtagctcagnccrtgnccraaytc GH3scree.r3(SEQ ID NO: 264) gtcaaagtggcggtagtcgatraanacnccytc GH3scree.r4(SEQ ID NO: 265) ggtgggcgagttgccgacggggttgactctgccrtanar GH3scree.r5(SEQ ID NO: 266) gccgggcagaccggcccagaggatggcngtnacrttngg GH3scree.r6(SEQ ID NO: 267) caggacggggccaaccgagtgaatgacnacdatngtrtt

PCR screening of Penicillium oxalicum strain IBT5387 genomic DNA wasperformed with two successive PCRs. Each forward primers (f1, f2, f5,and f6) (0.33 μl of a 10 mM stock) was combined with each reverseprimers (r1, r2, r3, r4, r5, and r6) (0.33 μl of a 10 mM stock) in a 10μl mix containing 0.33 μl genomic DNA, 5 μl of Reddy Mix Extensor PCRMaster Mix 1 (Cat #AB-0794/A, ABgene UK, commercialized by Thermo FisherScientific). A hot-start PCR reaction was carried out in a DYAD® PCRmachine for 2 minutes at 94° C. followed by 9 cycles each at 94° C. for15 seconds, 63° C. for 30 seconds with a 1° C. decrease for each cycle,68° C. for 1 minute 45 seconds, and followed by 24 cycles each at 94° C.for 15 seconds, 55° C. for 30 seconds, 68° C. for 1 minute 45 seconds,supplemented by a 7 minutes extension at 68° C. PCR-products producedduring this first PCR were re-amplified with their corresponding primersby transferring 0.5 μl of the first PCR reaction to a second 20 μl mixcontaining the same concentration of primers, dNTPs, polymerase, andbuffer than in the first PCR reaction. The second PCR was carried out onthe same PCR block at 94° C. for 2 minutes followed by 24 cycles each at94° C. for 15 seconds, 58° C. for 30 seconds, 68° C. for 1 minute 45seconds and completed by a 7 minutes extension at 68° C. PCR productsproduced during the second amplification were analyzed on 1% agarose gelelectrophoresis in TAE buffer. Single bands ranging from 2000 to 800 ntsin size were collected and eluted using the GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. Purified DNAsamples were directly sequenced with primers used for amplification.

Sequences were assembled in SeqMan v7.2.1 (DNA star, Madison, Wis., USA)into a contig that was used for nblast searches (Altschul et al., 1997,Nucleic Acids Res. 25: 3389-3402) on publicly available sequences.Results of the search indicated that the sequence GenBank EU700488.1 wasnearly identical to the contig identified. Therefore, the cloningprimers 5786GH3-50f and 5786GH3-50r were designed based on the sequenceinformation of the cds from GenBank EU700488.1 for IN-FUSION™ cloningaccording to the manufacturer instructions for cloning in the expressionvector pDAu109 (WO 2005/042735).

5786GH3-50f1: (SEQ ID NO: 268)5′-acacaactggggatccaccATGAAGCTCGAGTGGCTGGAAGC-3′ 5786GH3-50r1(SEQ ID NO: 269) 5′-agatctcgagaagcttaCTGCACCTTGGGCAGATCGGCTG-3′

The Penicillium oxalicum beta-glucosidase gene was amplified by PCRusing the two cloning primers described previously in a PCR reactionthat was performed with genomic DNA prepared from the strain IBT5387(from DTU received in 1992). The PCR reaction was composed of 1 μl ofgenomic DNA, 0.75 μl of primer 5786GH3-51f1 (10 μM); 0.75 μl of primer5786GH3-51r1 (10 μM); 3 μl of 5×HF buffer, 0.25 μl of 50 mM MgCl₂, 0.3μl of 10 mM dNTP; 0.15 μl of PHUSION® DNA polymerase, and PCR-gradewater up to 15 μl. The PCR reaction was performed using a DYAD® PCRmachine programmed for 2 minutes at 98° C. followed by 10 touchdowncycles at 98° C. for 15 seconds, 70° C. (−1° C./cycle) for 30 seconds,and 72° C. for 2 minutes 30 seconds; and 25 cycles each at 98° C. for 15seconds, 60° C. for 30 seconds, 72° C. for 2 minutes 30 seconds, and 5minutes at 72° C.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.8 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Penicillium oxalicum beta-glucosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an I N-FUSION™ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Penicillium oxalicumbeta-glucosidase gene sequence was verified by Sanger sequencing beforeheterologous expression.

The coding sequence is 2964 bp including the stop codon and isinterrupted by 5 introns of 101 bp (nucleotides 61 to 161), 64 bp(nucleotides 302 to 365), 79 bp (nucleotides 411 to 489), 63 bp(nucleotides 543 to 605), and 71 bp (nucleotides 2660 to 2730). Theencoded predicted protein is 861 amino acids. Using the SignalP program(Nielsen et al., 1997, Protein Engineering 10: 1-6), a signal peptide of19 residues was predicted. The predicted mature protein contains 842amino acids.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 were added and gently mixed. The mixture was incubated at 37° C. for30 minutes and the protoplasts were spread on COVE sucrose (1 M) platessupplemented with 10 mM acetamide and 15 mM CsCl for transformantselection. After incubation for 4-7 days at 37° C. spores of severaltransformants were seeded on YP-2% maltodextrin medium. After 4 dayscultivation at 30° C. culture broth was analyzed in order to identifythe best transformants based on their ability to produce a large amountof active Penicillium oxalicum beta-glucosidase. The screening was basedon intensity of the band corresponding to the heterologous expressedprotein determined by SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-glucopyranoside (pNPG) as described in Example 16.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then carried out in 250 ml shake flasks using DAP-2C-1 medium for 4days at 30° C. with shaking at 100 rpm.

Filtered broth was concentrated and washed with deionized water using atangential flow concentrator equipped with a Sartocon® Slice cassettewith a 10 kDa MW-CO polyethersulfone membrane (Sartorius Stedim BiotechGmbH, Goettingen, Germany). The concentrate was added to M ammoniumsulphate and loaded onto a Phenyl Toyopearl (650M) column (TosohCorporation, 3-8-2 Shiba, Minato-ku, Tokyo, Japan) equilibrated in 2 Mammonium sulphate, and bound proteins were eluted with 1 M ammoniumsulphate. The eluted protein was buffer exchanged with 25 mM HEPES pH7.5 with sodium chloride by ultrafiltration with a 10 kDapolyethersulfone membrane using Vivaspin 20 (Sartorius Stedim BiotechGmbH, Goettingen, Germany). Protein concentration was determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 79: Preparation of Talaromyces emersonii Strain CBS 549.92 GH3Beta-Glucosidase

The Talaromyces emersonii strain CBS 549.92 GH3 beta-glucosidase (SEQ IDNO: 189 [DNA sequence] and SEQ ID NO: 190 [deduced amino acid sequence])was obtained according to the procedure described below.

Genomic DNA was isolated according to the procedure described in Example73.

The Talaromyces emersonii beta-glucosidase gene was isolated by PCRusing two cloning primers GH3-11f and GH3-11r shown below, which weredesigned based on the publicly available Talaromyces emersoniifull-length sequence (Genbank AY072918.4) for direct cloning using theIN-FUSION™ strategy.

Primer GH3-11f: (SEQ ID NO: 270)acacaactggggatccaccatgaggaacgggttgctcaaggtcg Primer GH3-11r:(SEQ ID NO: 271) agatctcgagaagcttaaattccagggtatggcttaaggggc

A PCR reaction was performed with genomic DNA prepared from Talaromycesemersonii strain CBS 549.92 in order to amplify the full-length gene.The PCR reaction was composed of 1 μl of genomic DNA, 0.75 μl of primerGH3-11f (10 μM); 0.75 μl of primer GH3-11r (10 μM); 3 μl of 5×HF buffer,0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP; 0.15 μl of PHUSION® DNApolymerase, and PCR-grade water up to 15 μl. The PCR reaction wasperformed using a DYAD® PCR machine programmed for 2 minutes at 98° C.followed by 10 touchdown cycles at 98° C. for 15 seconds, 70° C. (−1°C./cycle) for 30 seconds, and 72° C. for 2 minutes 30 seconds; and 25cycles each at 98° C. for 15 seconds, 60° C. for 30 seconds, 72° C. for2 minutes 30 seconds, and 5 minutes at 72° C.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.9 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Talaromyces emersonii beta-glucosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an I N-FUSION™ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Talaromyces emersoniibeta-glucosidase gene sequence was verified by Sanger sequencing beforeheterologous expression.

The coding sequence is 2925 bp including the stop codon and isinterrupted by 6 introns of 60 bp (nucleotides 61 to 120), 61 bp(nucleotides 261 to 321), 60 bp (nucleotides 367 to 426), 57 bp(nucleotides 480 to 536), 56 bp (nucleotides 1717 to 1772), and 54 bp(nucleotides 2632 to 2685). The encoded predicted protein is 858 aminoacids. Using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6), a signal peptide of 19 residues was predicted. Thepredicted mature protein contains 839 amino acids.

Protoplasts of Aspergillus oryzae BECh2 (WO 2000/39322) were prepared asdescribed in WO 95/02043. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 were added and gently mixed. The mixture was incubated at 37° C. for30 minutes and the protoplasts were spread on COVE sucrose (1 M) platessupplemented with 10 mM acetamide and 15 mM CsCl for transformantselection. After incubation for 4-7 days at 37° C. spores of severaltransformants were seeded on YP-2% maltodextrin medium. After 4 dayscultivation at 30° C. culture broth was analyzed in order to identifythe best transformants based on their ability to produce a large amountof active Talaromyces emersonii beta-glucosidase. The screening wasbased on intensity of the band corresponding to the heterologousexpressed protein determined by SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-glucopyranoside (pNPG) as described in Example 16.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then carried out in 250 ml shake flasks using YP-2% maltodextrinmedium for 4 days at 30° C. with shaking at 100 rpm. The broth wasfiltered using standard methods. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 80: Preparation of Thermoascus crustaceus Strain CBS 181.67GH61A Polypeptide

The Thermoascus crustaceus strain CBS 181.67 GH61A polypeptide havingcellulolytic enhancing activity (SEQ ID NO: 191 [DNA sequence] and SEQID NO: 192 [deduced amino acid sequence]) was obtained according to theprocedure described below.

Aspergillus oryzae strain HowB101 (WO 95/35385) was used as a host forrecombinantly expressing the Thermoascus crustaceus GH61 polypeptideshaving cellulolytic enhancing activity.

Thermoascus crustaceus strain CBS 181.67 was inoculated onto a PDA plateand incubated for 3-4 days at 45° C. in the darkness. Severalmycelia-PDA plugs were inoculated into 500 ml shake flasks containing100 ml of NNCYP-PCS medium. The flasks were incubated for 6 days at 45°C. with shaking at 160 rpm. The mycelia were collected at day 3, day 4,day 5, and day 6. Then the mycelia from each day were combined andfrozen in liquid nitrogen, and then stored in a −80° C. freezer untiluse.

Genomic DNA was extracted using a DNEASY® Plant Mini Kit (QIAGEN Inc.,Valencia, Calif., USA). Total RNA was isolated by using a RNEASY® PlantMini Kit. cDNA was synthesized by following the instructions of the 3′Rapid Amplification of cDNA End System (3′ RACE) (InvitrogenCorporation, Carlsbad, Calif., USA).

Four degenerate primers shown below were designed based on conservedregions of known GH61 sequences.

GH61A scF1: (SEQ ID NO: 272) 5′-GCNACNGAYCTNGGNTTTG-3′ GH61A scF2:(SEQ ID NO: 273) 5′-GCNACNGAYCTNGGNTTCG-3′ GH61A scF3: (SEQ ID NO: 274)5′-GCNACNGAYTTRGGNTTYG-3′ GH61A scR1: (SEQ ID NO: 275)5′-CAYTGNGGRTARTTYTGNGC-3′

PCR was performed by using a combination of forward primers GH61A scF1,GH61 scF2, and GH61A scF3, and reverse primer GH61A scR1 and cDNA astemplate. The amplification reaction was composed of 5 μl of 10×PCRbuffer (Invitrogen Corporation, Carlsbad, Calif., USA), 2 μl of 25 mMMgCl₂, 1 μl of 10 mM dNTP, 1 μl of 100 μM forward primer, 1 μl of 100 μMreverse primer, 2 μl of cDNA, 0.5 μl of Taq DNA polymerase High Fidelity(Invitrogen Corporation, Carlsbad, Calif., USA), and 37.5 μl of H₂O. Theamplification was performed using an Peltier Thermal Cycler programmedfor denaturing at 94° C. for 2 minutes; 30 cycles each at 94° C. for 40seconds, 50° C. for 40 seconds, and 72° C. for 1 minute; and a finalextension at 72° C. for 10 minutes.

A PCR product of approximately 500 base pairs was detected by 1% agarosegel electrophoresis using TBE buffer. The PCR fragment was excised fromthe gel and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions, directlysequenced, and confirmed to be a GH61A partial gene by blast. Based onthis partial sequence, new primers shown below were designed for 5′ and3′ end cloning using a Genome Walking Kit (Takara Bio Inc., Otsu, Shiga,Japan):

61ASPR1: (SEQ ID NO: 276) 5′-TGCAAGGAGCAAGGTAGTTGA-3′ 61ASPR2:(SEQ ID NO: 277) 5′-GAGTCCATTCCAGCTTGACGGT-3′ 61ASPF1: (SEQ ID NO: 278)5′-TCAGACAATCTGATAGCGGC-3′ 61ASPF2: (SEQ ID NO: 279)5′-ATCCCAACCACAACTGCACCT-3′

For 5′ end and 3′ end cloning, the primary amplifications were composedof 2 μl of genomic DNA as template, 2.5 mM each of dATP, dTTP, dGTP, anddCTP, 100 μmol of AP2 (provided by the Genome Walking Kit) and 10 μmolof primer 61ASPR1 for 5′ end cloning or 100 μmol of AP3 (provided by theGenome Walking Kit), and 10 μmol of primer 61ASPF1 for 3′ end cloning, 5μl of 10×LA PCR Buffer II (provided by the Genome Walking Kit), and 2.5units of TakaRa LA Taq DNA polymerase (provided by the Genome WalkingKit) in a final volume of 50 μl. The amplifications were performed usingan Peltier Thermal Cycler programmed for pre-denaturing at 94° C. for 1minute and 98° C. for 1 minute; five cycles each at a denaturingtemperature of 94° C. for 30 seconds; annealing at 60° C. for 1 minuteand elongation at 72° C. for 2 minutes; 1 cycle of denaturing at 94° C.for 30 seconds; annealing at 25° C. for 3 minutes and elongation at 72°C. for 2 minutes; fifteen repeats of 2 cycles at 94° C. for 30 seconds,62° C. for 1 minutes, and 72° C. for 2 minutes; followed by 1 cycle at94° C. for 30 seconds, 44° C. for 1 minutes, and 72° C. for 2 minutes;and a final extension at 72° C. for 10 minutes. The heat block then wentto a 4° C. soak cycle.

The secondary amplifications were composed of 2 μl of 20× dilutedprimary PCR product as templates, 2.5 mM each of dATP, dTTP, dGTP, anddCTP, 100 μmol of AP2, and 10 pmol of primer 61ASPR2 for 5′ end cloningor 100 μmol of AP3 and 10 μmol of primer 61ASPF2 for 3′ end cloning, 5μl of 10×LA PCR Buffer II, and 2.5 units of TakaRa LA Taq DNA polymerasein a final volume of 50 μl. The amplifications were performed using anPeltier Thermal Cycler programmed for fifteen repeats of 2 cycles of 94°C. for 30 seconds; 62° C. for 1 minutes; 72° C. for 2 minutes; followedby 1 cycle at 94° C. for 30 seconds, 44° C. for 1 minutes, and 72° C.for 2 minutes; and a final extension at 72° C. for 10 minutes. The heatblock then went to a 4° C. soak cycle.

The PCR products from the 5′ and 3′ end PCR were recovered andsequenced. They were identified as the 5′ end and 3′ end of the GH61Apolypeptide gene. Then the three sequences including the partial gene,5′ end, and 3′ end were assembled to generate the full-length GH61A.

The obtained full-length gene showed that the sequence contains a codingregion of 871 nucleotides including 1 intron and stop codon, and encodes251 amino acids with a predicted signal peptide of 22 amino acids.

Based on the full-length Thermoascus crustaceus GH61A gene sequence,oligonucleotide primers, shown below, were designed to amplify the GH61Agene from genomic DNA of Thermoascus crustaceus CBS 181.67. AnIN-FUSION® CF Dry-Down Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 280)5′-ACACAACTGGGGATCCACCATGGCCTTTTCCCAGATAATGGCTA-3′ Antisense primer:(SEQ ID NO: 281) 5′-GTCACCCTCTAGATCTGGATCGCAGGAGCGTTCAGA-3′Bold letters represent the coding sequence for the sense primer and thedownstream sequence of the stop codon for the antisense primer. Theremaining sequence is homologous to the insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Thermoascus crustaceus genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 2 μl of 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 1 unit of PHUSION™ High-Fidelity DNA Polymerase in a finalvolume of 50 μl. The amplification was performed using a Peltier ThermalCycler programmed for denaturing at 98° C. for 1 minute; 5 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 70° C. for 30 seconds,with a 1° C. decrease per cycle, and elongation at 72° C. for 30seconds; 25 cycles each at 98° C. for 15 seconds and 72° C. for 90seconds; and a final extension at 72° C. for 10 minutes. The heat blockthen went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.0 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn HI and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION® CF Dry-Down PCR Cloning Kit resulting in pGH61a51486 in whichtranscription of the Thermoascus crustaceus GH61A gene was under thecontrol of the Aspergillus oryzae TAKA-alpha-amylase promoter. In brief,30 ng of pPFJO355 digested with Barn HI and Bgl II and 50 ng of theThermoascus crustaceus GH61A gene purified PCR product were added to areaction vial and resuspended in a final volume of 10 μl with deionizedwater. The reaction was incubated at 37° C. for 15 minutes and then 50°C. for 15 minutes. Three μl of the reaction were used to transform E.coli TOP10 competent cells according to the manufacturer's instructions.The transformation was spread on LB plates supplemented with 100 μg ofampicillin per ml and incubated at at 37° C. for 1 day. An E. colitransformant containing a plasmid designated pGH61a51486 was detected bycolony PCR and plasmid DNA was prepared using a QIAprep Spin MiniprepKit (QIAGEN Inc., Valencia, Calif., USA). The Thermoascus crustaceusGH61A gene insert in pGH61a51486 was confirmed by DNA sequencing using a3730XL DNA Analyzer.

The same PCR fragment was cloned into pGEM-T vector using a pGEM-TVector System to generate pGEM-T-GH61a51486. The Thermoascus crustaceusGH61A gene insert in pGEM-T-GH61a51486 was confirmed by DNA sequencingusing a 3730XL DNA Analyzer. E. coli strain T-51486A, designatedNN059126, containing pGEM-T-GH61a51486, was deposited at the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), MascheroderBraunschweig, Germany, on Jun. 10, 2009, and assigned accession numberDSM 22656.

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The nucleotide sequence and deduced amino acid sequence of theThermoascus crustaceus gh61a gene are shown in SEQ ID NO: 191 and SEQ IDNO: 192, respectively. The coding sequence is 871 bp including the stopcodon and is interrupted by one intron of 115 base pairs (nucleotides105-219). The encoded predicted protein is 251 amino acids. The % G+Ccontent of the full-length coding sequence and the mature codingsequence are 50.23% and 52.55%, respectively. Using the SignalP softwareprogram (Nielsen et al., 1997, Protein Engineering 10: 1-6), a signalpeptide of 22 residues was predicted. The predicted mature proteincontains 229 amino acids with a predicted molecular mass of 26.35 kDa.

Aspergillus oryzae HowB101 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422 andtransformed with 3 μg of pGH61a51486. The transformation yielded about50 transformants. Twelve transformants were isolated to individualMinimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C. with shaking at 150 rpm. After 3days incubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT BLUE™ (Expedeon Ltd., Babraham Cambridge, UK).SDS-PAGE profiles of the cultures showed that the majority of thetransformants had a major band of approximately 30 kDa. The expressionstrain was designated Aspergillus oryzae EXP03151.

A slant of Aspergillus oryzae EXP03151 was washed with 10 ml of YPMmedium and inoculated into a 2 liter flask containing 400 ml of YPMmedium to generate broth for characterization of the enzyme. The culturewas harvested on day 3 and filtered using a 0.45 μm DURAPORE® Membrane(Millipore, Bedford, Mass., USA).

The filtered broth was concentrated and buffer exchanged using atangential flow concentrator equipped with a Sartocon® Slice cassettewith a 10 kDa cut-off polyethersulfone membrane (Sartorius StedimBiotech GmbH, Goettingen, Germany) with 25 mM HEPES, pH 7.0. The proteinwas applied to a Q Sepharose™ Fast Flow column (GE Healthcare,Piscataway, N.J., USA) equilibrated in 25 mM HEPES pH 7.0. The proteinwas recovered in the eluate. Protein concentration was determined usinga Microplate BCA™ Protein Assay Kit in which bovine serum albumin wasused as a protein standard.

Example 81: Preparation of Talaromyces emersonii Strain NN05002 GH10Xylanase

The Talaromyces emersonii strain NN05002 GH10 xylanase (SEQ ID NO: 193[DNA sequence] and SEQ ID NO: 194 [deduced amino acid sequence]) wasobtained according to the procedure described below.

Talaromyces emersonii was grown on a PDA agar plate at 45° C. for 3days. Mycelia were collected directly from the agar plate into asterilized mortar and frozen under liquid nitrogen. Frozen mycelia wereground, by mortar and pestle, to a fine powder, and genomic DNA wasisolated using a DNeasy® Plant Mini Kit.

Oligonucleotide primers, shown below, were designed to amplify the GH10xylanase gene from genomic DNA of Talaromyces emersonii. An IN-FUSION™CF Dry-down Cloning Kit was used to clone the fragment directly into theexpression vector pPFJO355, without the need for restriction digestionand ligation.

Sense primer: (SEQ ID NO: 282)5′-ACACAACTGGGGATCCACCATGGTTCGCCTCAGTCCAG-3′ Antisense primer:(SEQ ID NO: 283) 5′-GTCACCCTCTAGATCTTTACAGACACTGCGAGTAATACTCATTG-3′Bold letters represented the coding sequence. The remaining sequence washomologous to the insertion sites of pPFJO355.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Talaromyces emersonii genomic DNA, 10 μl of 5×GCBuffer, 1.5 μl of DMSO, 5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6unit of Phusion™ High-Fidelity DNA Polymerase in a final volume of 50μl. The amplification was performed using a Peltier Thermal Cyclerprogrammed for denaturing at 98° C. for 40 seconds; 8 cycles ofdenaturing at 98° C. for 15 seconds, annealing at 65° C. for 30 seconds,with a 1° C. decrease per cycle and elongation at 72° C. for 80 seconds;and another 23 cycles each at 98° C. for 15 seconds, 58° C. for 30seconds and 72° C. for 80 seconds; final extension at 72° C. for 7minutes. The heat block then went to a 10° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 1.4 kb product band was excisedfrom the gel, and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA® GFX® PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ CF Dry-down PCR Cloning resulting in pxynTe50022 in whichtranscription of the Talaromyces emersonii GH10 xylanase gene was underthe control of the Aspergillus oryzae TAKA amylase promoter. In brief,20 ng of pPFJO355 digested with Barn I and Bgl II, and 60 ng of theTalaromyces emersonii GH10 xylanase gene purified PCR product were addedto a reaction vial and resuspended in a final volume of 10 μl withaddition of deionized water. The reaction was incubated at 37° C. for 15minutes and then 50° C. for 15 minutes. Three μl of the reaction wereused to transform E. coli TOP10 competent cells. An E. coli transformantcontaining pxynTe50022 was detected by colony PCR and plasmid DNA wasprepared using a QIAprep Spin Miniprep Kit. The Talaromyces emersoniiGH10 xylanase gene insert in pxynTe50022 was confirmed by DNA sequencingusing a 3730XL DNA Analyzer.

Aspergillus oryzae HowB101 (WO 95/35385) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422 and transformed with 3 μg of pxynTe50022. The transformationyielded about 50 transformants. Twelve transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NUPAGE® NOVEX® 4-12% Bis-Tris Gel with MES bufferaccording to the manufacturer's instructions. The resulting gel wasstained with INSTANT® Blue. SDS-PAGE profiles of the cultures showedthat the majority of the transformants had a major smeary band ofapproximately 55 kDa. The expression strain was designated as A. oryzaeEXP03373.

A slant of A. oryzae EXP03373 was washed with 10 ml of YPM medium andinoculated into six 2 liter flasks, each containing 400 ml of YPMmedium, to generate broth for characterization of the enzyme. Theculture was harvested on day 3 by filtering the culture throughMIRACLOTH® (CALBIOCHEM, Inc. La Jolla, Calif., USA). The filteredculture broth was then again filtered using a 0.45 μm DURAPORE Membrane.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit in which bovine serum albumin was used as a protein standard.

A 1600 ml volume of filtered broth supernatant of A. oryzae EXP03373 wasprecipitated with ammonium sulfate (80% saturation), re-dissolved in 100ml of 25 mM Bis-Tris pH 6.0, dialyzed against the same buffer, andfiltered through a 0.45 μm filter; the final volume was 200 ml. Thesolution was applied to a 40 ml Q Sepharose® Fast Flow columnequilibrated with 25 mM Bis-Tris pH 6.0, and the proteins were elutedwith a linear NaCl gradient (0-0.4 M). Fractions with activity againstAZCL-xylan were analyzed by SDS-PAGE using a NUPAGE® NOVEX® 4-12%Bis-Tris Gel with MES buffer. Fractions with the correct molecularweight were pooled and concentrated by ultra filtration.

The supernatants were tested for endocellulase activity by microtiterplate assay as described below. A solution of 0.2% of the blue substrateAZCL-Xylan (Megazyme) was suspended in a 0.1 M sodium acetate buffer (pH5.5) under stirring. The solution was distributed under stirring to amicrotiter plate (200 μl to each well). Twenty μl of enzyme sample wasadded and incubated in an EPPENDORF® Thermomixer for 20 minutes at 50°C. and 650 rpm. A denatured enzyme sample (100° C. boiling for 20minutes) was used as blank. After incubation the colored solution wasseparated from the solid by centrifugation at 3000 rpm for 5 minutes at4° C. Then 150 μl of supernatant was transferred to a microtiter plateand the absorbance was measured using a Spectra Max M2 at 595 nm.

Example 82: Preparation of Penicillium sp. Strain NN51602 GH10 Xylanase

The Penicillium sp. strain NN51602 GH10 xylanase (SEQ ID NO: 195 [DNAsequence] and SEQ ID NO: 196 [deduced amino acid sequence]) was obtainedaccording to PCT/US10/032034. Protein concentration was determined usinga Microplate BCA™ Protein Assay Kit in which bovine serum albumin wasused as a protein standard.

Example 83: Preparation of Meripilus giganteus strain CBS 521.95 GH10xylanase

The Meripilus giganteus strain CBS 521.95 GH10 xylanase (SEQ ID NO: 197[DNA sequence] and SEQ ID NO: 198 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 97/27290 except Aspergillusoryzae Bech2 (WO 2000/39322) was used as a host and pDAu75 as theexpression vector. Vector pDAu75 is a derivative of pJa1721 (WO03/008575). Plasmid pDAu75 mainly differs from pJa1721 in that theselection marker URA3 of E. co/i has been dirupted by the insertion ofthe ampicillin resistance gene E. co/i beta lactamase, which allows forrapid selection of positive recombinant E. co/i clones usingcommercially available and highly competent strains on commonly used LBampicillin plates. The ampicillin resistance gene is entirely removableusing the two flanking Not I sites restoring a functional selectionmarker URA3. The techniques used for making pDAu75 from pJa1721 arecommon molecular biology techniques for DNA cloning. Cloning of the cDNAsequence of the Meripilus giganteus GH10 xylanase gene into pDAu75 wasperformed by a restriction/ligation cloning procedure from the xylanaseproducing yeast colony as described in WO 97/27290.

The broth was filtered using Whatmann glass filter GF/D, GF/A, GF/C,GF/F (2.7 μm, 1.6 μm, 1.2 μm and 0.7 μm, respectively) followed byfiltration through a 0.45 μm filter.

Ammonia sulfate was added to the filtered broth to a final concentrationof 3 M and the precipitate was collected after centrifugation at10,000×g for 30 minutes. The precipitate was dissolved in 10 mM Tris/HClpH 8.0 and dialyzed against 10 mM Tris/HCl pH 8.0 overnight. Thedialyzed preparation was applied to a 150 ml Q SEPHAROSE® Fast Flowcolumn equilibrated with 10 mM Tris/HCl pH 8.0 and the enzyme was elutedwith a 1050 ml (7 column volumes) linear salt gradient from 0 to 1 MNaCl in 10 mM Tris/HCl pH 8.0. Elution was followed with A280 nmdetection and fractions were collected and assayed for xylanase activityusing 0.2% AZCL-Arabinoxylan from wheat (Megazyme) in 0.2 M sodiumphosphate pH 6.0 plus 0.01% TRITON® X100. Fractions containing xylanaseactivity were pooled and stored at −20° C. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 84: Preparation of Dictyoglomus thermophilum Strain ATCC 35947GH11 Xylanase

The Dictyoglomus thermophilum GH11 xylanase (SEQ ID NO: 199 [DNAsequence] and SEQ ID NO: 200 [deduced amino acid sequence]) wasrecombinantly prepared according to the following procedure.

Bacillus subtilis strains were made competent using the method describedby Anagnostopoulos and Spizizen, 1961, Journal of Bacteriology 81:741-746. DNA sequencing was conducted with an ABI 3700 Sequencing(Applied Biosystems, Inc., Foster City, Calif., USA).

Bacillus subtilis strain SM025 was constructed as described below todelete an intracellular serine protease (ispA) gene in Bacillus subtilisstrain A164410 (Bindel-Connelly et al., 2004, J. Bacteriol. 186:4159-4167).

A deletion plasmid, pNNB194-ispAΔ, was constructed by splicing byoverlap extension (SOE) (Horton et al., 1989, Gene 77: 61-8). FlankingDNA sequences 5′ and 3′ of the ispA gene were obtained by PCRamplification from chromosomal DNA derived from Bacillus subtilis strain164Δ5 (U.S. Pat. No. 5,891,701) using primer pairs 994525/994526 and994527/994528, respectively, shown below. Chromosomal DNA was obtainedaccording to the procedure of Pitcher et al., 1989, Lett. Appl.Microbiol. 8: 151-156.

Primer 994525: (SEQ ID NO: 284) 5′-GGATCCATTATGTAGGGCGTAAAGC-3′Primer 994526: (SEQ ID NO: 285) 5′-TTAGCAAGCTTAATCACTTTAATGCCCTCAG-3′Primer 994527: (SEQ ID NO: 286) 5′-TGATTAAGCTTGCTAATCCGCAGGACACTTC-3′Primer 994528: (SEQ ID NO: 287) 5′-GGTACCAACACTGCCTCTCTCATCTC-3′

PCR amplifications were conducted in 50 μl reactions composed of 10 ngof Bacillus subtilis strain 164Δ5 chromosomal DNA, 0.4 μM of eachprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II(Applied Biosystems, Inc., Foster City, Calif., USA) with 2.5 mM MgCl₂,and 2.5 units of AmpliTaq GOLD® DNA Polymerase (Applied Biosystems,Inc., Foster City, Calif., USA). The reactions were performed in aROBOCYCLER® 40 Temperature Cycler (Stratagene, Corp., La Jolla, Calif.,USA) programmed for 1 cycle at 95° C. for 10 minutes; 25 cycles each at95° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 1 minute; and 1cycle at 72° C. for 7 minutes.

The PCR products were resolved by 0.8% agarose gel electrophoresis using0.5×TBE buffer (50 mM Tris base-50 mM boric acid-1 mM disodium EDTA). Aband of approximately 400 bp obtained using the primer pair994525/994526 for the 5′ flanking DNA sequence of the ispA gene wasexcised from the gel and extracted using a QIAQUICK® Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA). A band of approximately 400 bpobtained using the primer pair 994527/994528 for the 3′ flanking DNAsequence of the ispA gene was excised from the gel and extracted using aQIAQUICK® Gel Extraction Kit.

The final SOE fragment was amplified using the same procedure above withthe 400 bp fragments as templates and primers 994525 and 994528, shownabove, to produce an ispA deletion fragment. The PCR product ofapproximately 800 kb was resolved by 0.8% agarose gel electrophoresisusing 0.5×TBE buffer.

The final 800 kb SOE fragment was cloned into pCR®2.1 (Invitrogen,Carlsbad, Calif., USA) using a TA-TOPO® Cloning Kit (Invitrogen,Carlsbad, Calif., USA) and transformed into ONE SHOT® TOP10 ChemicallyCompetent E. coli cells (Invitrogen, Carlsbad, Calif., USA) according tothe manufacturer's instructions. Transformants were selected on 2×YTagar plates supplemented with 100 μg of ampicillin per ml and incubatedat 37° C. for 16 hours. The DNA sequence of the cloned fragment wasverified by DNA sequencing with M13 forward and reverse primers(Invitrogen, Inc, Carlsbad, Calif., USA). The plasmid was designatedpCR®2.1-ispAΔ.

Plasmid pCR2.1-ispAΔ was digested with Barn HI and Asp718 and subjectedto 0.8% agarose gel electrophoresis using 0.5×TBE buffer to isolate theispA deletion fragment. A 800 bp fragment corresponding to the ispAdeletion fragment was excised from the gel and extracted using aQIAQUICK® Gel Extraction Kit.

The temperature sensitive plasmid pNNB194 (pSK+/pE194; U.S. Pat. No.5,958,728) was digested with Barn HI and Asp718 and resolved by 0.8%agarose gel electrophoresis using 0.5×TBE buffer to isolate the vectorfragment. A 6600 bp vector fragment of pNNB194 was excised from the geland extracted using a QIAQUICK® Gel Extraction Kit.

The ispA deletion fragment and the pNNB194 fragment were ligatedtogether using a Rapid DNA Ligation Kit (Roche Applied Science,Indianapolis, Ind., USA) and the ligation mix was transformed into E.coli SURE® cells (Stratagene Corp., La Jolla, Calif., USA) selecting forampicillin resistance according to the manufacturer's instructions.Plasmid DNA was isolated from eight transformants using a BIOROBOT®9600, digested with Barn HI and Asp718, and analyzed by agaroseelectrophoresis as described above to identify plasmids which harboredthe ispAΔ fragment. One transformant was identified and designatedpNNB194-ispAΔ.

Plasmid pNNB194-ispAΔ was introduced into Bacillus subtilis A164410(Bindel-Connelly et al., 2004, J. Bacteriol. 186: 4159-4167) andintegrated at the ispA locus by selective growth at 45° C. on Tryptoseblood agar base (TBAB) plates supplemented with 1 μg of erythromycin and25 μg of lincomycin per ml. The integrated plasmid was then excised bynon-selective growth on LB medium at 34° C. Chromosomal DNA was isolatedfrom several erythromycin sensitive clones according to the method ofPitcher et al., 1989, supra, and analyzed by PCR using primers 994525and 994528 using the same method above to confirm the presence of theispA deletion. One such clone was designated Bacillus subtilis SMO25.

A linear integration vector-system was used for the expression cloningof a synthetic Dictyoglomus thermophilum Family 11 xylanase gene withouta binding domain and without a signal peptide. The synthetic genesequence was based on the public gene sequence UNIPROT: P77853. Thesynthetic gene was codon optimized for expression in Bacillus subtilisfollowing recommendations by Gustafsson et al., 2004, Trends inBiotechnology 22: 346-353. The synthetic gene was generated by DNA2.0(Menlo Park, Calif., USA) and delivered as a cloned fragment in theirstandard cloning vector (kanamycin resistant). The xylanase gene wascloned as a truncated gene without binding domain and with the signalpeptide from Bacillus clausii serine protease gene (aprH, SAVINASE™,Novo Nordisk A/S, Bagsvrd, Denmark) (included in the flanking region).The gene was designed to contain a C-terminal HQHQHQHQP tag to easepurification. The forward primer was designed so the gene was amplifiedfrom the signal peptide cleavage site and it had 26 bases overhang(shown in italic in the table below). This overhang was complementary topart of one of the two linear vector fragments and was used when the PCRfragment and the vector fragments were assembled (described below). Thereverse primer was designed to amplify the truncated version of the geneand contained an overhang consisting of 30 bp encoding a HQHQHQHQP-tagand a stop codon (the overhang is shown in italic in the table below).This overhang was complementary to part of one of the two linear vectorfragments and was used when the PCR fragment and the vector fragmentswere assembled (described below).

The linear integration construct was a PCR fusion product made by fusionof each gene between two Bacillus subtils homologous chromosomal regionsalong with a strong promoter and a chloramphenicol resistance marker.The fusion was made by splicing by overlap extension (SOE) (Horton etal., 1989, supra). The SOE PCR method is also described in WO2003/095658. Each gene was expressed under the control of a triplepromoter system (described in WO 99/43835), consisting of the promotersfrom Bacillus licheniformis alpha-amylase gene (amyL), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis cryIIIA promoter including the mRNA stabilizing sequence.The gene coding for chloramphenicol acetyl-transferase was used asmarker (described, for example, by Diderichsen et al., 1993, Plasmid 30:312). The final gene construct was integrated by homologousrecombination into the pectate lyase locus of the Bacillus chromosome.

The GH11 xylanase gene was amplified from plasmid 7587 by PCR using theprimers shown in the Table below 1 below. The plasmid 7587 contains thesynthetic Dictyoglomus thermophilum GH11 xylanase gene (SEQ ID NO: 304for the DNA sequence and SEQ ID NO: 305 for the deduced amino acidsequence) without a binding domain and without a signal peptide.

Three fragments were PCR amplified to make the construct: the genefragment containing the truncated xylanase gene and the 26 bp and 30 bpflanking DNA sequences included in the primers as overhang, the upstreamflanking fragment (including a signal peptide from Savinase andamplified with the primers 260558 and iMB1361Uni2) and the downstreamflanking fragment (amplified with the primers 260559 and HQHQHQHQP-f).The flanking fragments were amplified from genomic DNA of the strainiMB1361 (described in patent application WO 2003/095658). All primersused are listed in the Table 1 below.

The gene fragment was amplified using a proofreading polymerase PHUSION™DNA Polymerase according to the manufacturer's instructions. The twoflanking DNA fragments were amplified with “Expand High Fidelity PCRSystem” (Roche-Applied-Science) according to standard procedures(following the manufacturer's recommendations). The PCR conditions wereas follows: 94° C. for 2 minutes followed by 10 cycles of (94° C. for 15seconds, 50° C. for 45 seconds, 68° C. for 4 minutes) followed by 20cycles of (94° C. for 15 seconds, 50° C. for 45 seconds, 68° C. for 4minutes (+20 seconds extension per cycle)) and ending with one cycle at68° C. for 10 min. The 3 PCR fragments were subjected to a subsequentSplicing by Overlap Extension (SOE) PCR reaction to assemble the 3fragments into one linear vector construct. This was pereformed bymixing the 3 fragments in equal molar ratios and a new PCR reaction wererun under the following conditions: initial 2 minutes. at 94° C.,followed by 10 cycles of (94° C. for 15 seconds, 50° C. for 45 seconds,68° C. for 5 minutes), 10 cycles of (94° C. for 15 seconds, 50° C. for45 seconds, 68° C. for 8 minutes), 15 cycles of (94° C. for 15 seconds,50° C. for 45 seconds, 68° C. for 8 minutes in addition 20 seconds extraper cycle). After the 1st cycle the two end primers 260558 and 260559were added (20 μMol of each). Two μl of the PCR product were transformedinto Bacillus subtilis. Transformants were selected on LB platessupplemented with 6 μg of chloramphenicol per ml. The truncated xylanaseconstruct was integrated by homologous recombination into the genome ofthe Bacillus subtilis host PL4250 (AprE−, NprE−, SrfC−, SpollAC−, AmyE−,comS+). One transformant, EXP01955, was selected for further work. Thexylanase coding region was sequenced in this transformant. It containedone mutation leading to a change of the HQHQHQHQP-tag to aHQHQHQHQQ-tag) but no other mutations were observed.

TABLE 1 Primers used Amplification of SPECIFIC PRIMER FORWARDSPECIFIC PRIMER REVERSE Truncated gene FORWARD (SEQ ID NO: 288)REVERSE (SEQ ID NO: 289) 5′-CTTTTAGTTCATCGATCGCATCGG5′-CTAGGGTTGATGCTGGTGTTGGTGC CTGCTCAGACATCAATCACACTTA-3′TGATGGCTGCCCTGAGAGAAAGTG-3′ Upstream flanking 260558: (SEQ ID NO: 290)iMB1361Uni2 (SEQ ID NO: 291) fragment 5′-GAGTATCGCCAGTAAGGGGCG-3′5′ AGCCGATGCGATCGATGAACTA 3′ Downstream flankingHQHQHQHQP-f (SEQ ID NO: 292) 260559: (SEQ ID NO: 293) fragment5′-CATCAGCACCAACACCAGCACCAG 5′-GCAGCCCTAAAATCGCATAAAGCCATAATCGCATGTTCAATCCGCTCCA C-3′ TA-3′

Chromosomal DNA from Bacillus subtilis strain EXP01955 was used as atemplate to PCR clone the Bacillus clausii serine protease gene (aprH,SAVINASE™, Novo Nordisk A/S, Bagsvrd, Denmark) signal sequence/mature D.thermophilum xylanase gene (CBM-deleted) into pCR2.1-TOPO using thefollowing primers which introduce a Sac I site at the 5′ end (justupstream of the aprH ribosome binding site) and a Mlu I site at the 3′end (just after the translation stop codon which was introduced afterthe Ser codon at position 691-693, thereby avoiding the incorporation ofthe HQHQHQHQQ-tag). Chromosomal DNA was obtained according to theprocedure of Pitcher et al., 1989, supra.

Primer 062405: (SEQ ID NO: 294)5′-GAGCTCTATAAAAATGAGGAGGGAACCGAATGAAGAAACC-3′ Primer 062406:(SEQ ID NO: 295) 5′-ACGCGTTTAGCTGCCCTGAGAGAAAGTG-3′

The PCR amplifications were conducted in 50 μl reactions composed of 10ng of B. subtilis EXP01955 chromosomal DNA, 0.4 μM of each primer, 200μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mMMgCl₂, and 2.5 units of AmpliTaq GOLD® DNA Polymerase. The reactionswere performed in a ROBOCYCLER® 40 Temperature Cycler programmed for 1cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute,50° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for7 minutes. A PCR product of approximately 740 kb of the truncatedxylanase gene was resolved by 0.8% agarose gel electrophoresis using0.5×TBE buffer, excised from the gel, and extracted using a QIAQUICK®Gel Extraction Kit.

The 740 kb fragment was cloned into pCR®2.1 using a TA-TOPO® Cloning Kitaccording to the manufacturer's instructions and transformed into ONESHOT® TOP10 Chemically Competent E. coli cells according to themanufacturer's instructions. Transformants were selected on 2×YT agarplates supplemented with 100 μg of ampicillin per ml and incubated at37° C. for 16 hours. The DNA sequence of the cloned fragment wasverified by DNA sequencing with M13 forward and reverse primers. Theplasmid was designated pCR2.1-Dt xyl.

DNA sequencing revealed that there was an extra G at position 19 of thesequence encoding the aprH signal sequence. A QUIKCHANGE® XLSite-Directed Mutagenesis Kit (Stratagene Corp., La Jolla, Calif., USA)was utilized to correct the mistake in plasmid pCR2.1-Dt xyl using thefollowing primers to delete the extra G residue:

Primer 062535: (SEQ ID NO: 296) 5′-CCGTTGGGGAAAATTGTCGC-3′Primer 062536: (SEQ ID NO: 297) 5′-GCGACAATTTTCCCCAACGG-3′The kit was used according to the manufacturer's instructions and thechange was successfully made resulting in plasmid pCR2.1-Dt xyl2.

Plasmid pCR2.1-Dt xyl2 and pMDT100 WO 2008/140615 were digested with SacI and Mlu I. The digestions were each resolved by 0.8% agarose gelelectrophoresis using 0.5×TBE buffer. A vector fragment of approximately8.0 kb from pMDT100 and a xylanase gene fragment of approximately 700 bpfrom pCR2.1-Dt xyl2 were excised from the gels and extracted using aQIAQUICK® Gel Extraction Kit. The two purified fragments were ligatedtogether using a Rapid DNA Ligation Kit.

Competent cells of Bacillus subtilis 16844 were transformed with theligation products according to the method of Young and Spizizen, 1961,Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson,1971, Journal of Molecular Biology 56: 209-221. Bacillus subtilis 16844is derived from the Bacillus subtilis type strain 168 (BGSC 1A1,Bacillus Genetic Stock Center, Columbus, Ohio, USA) and has deletions inthe spolIAC, aprE, nprE, and amyE genes. The deletion of the four geneswas performed essentially as described for Bacillus subtilis A164Δ5(U.S. Pat. No. 5,891,701).

Bacillus subtilis transformants were selected at 37° C. after 16 hoursof growth on TBAB plates supplemented with 5 μg of chloramphenicol perml. To screen for integration of the plasmid by double cross-over at theamyE locus, Bacillus subtilis primary transformants were patched ontoTBAB plates supplemented with 6 μg of neomycin per ml and onto TBABplates supplemented with 5 μg of chloramphenicol per ml. Integration ofthe plasmid by double cross-over at the amyE locus does not incorporatethe neomycin resistance gene and therefore renders the strain neomycinsensitive. A chloramphenicol resistant, neomycin sensitive transformantwas identified, which harbored the Dictyoglomus thermophilum xylanaseexpression cassette in the amyE locus, and designated Bacillus subtilis168 with pSMO271.

Genomic DNA was isolated from Bacillus subtilis 168 with pSMO271(Pitcheret al., 1989, supra) and 0.1 μg was transformed into competent Bacillussubtilis SM025. Transformants were selected on TBAB plates supplementedwith 5 μg of chloramphenicol per ml at 37° C. A chloramphenicolresistant transformant was single colony purified and designatedBacillus subtilis SM 047.

The Bacillus subtilis strain designated SM047 was streaked on agarslants and incubated for about 24 hours at 37° C. The agar medium wascomposed per liter of 10 g of soy peptone, 10 g of sucrose, 2 g oftrisodium citrate dihydrate, 4 g of KH₂PO₄, 5 g of Na₂HPO₄, 15 g ofBacto agar, 0.15 mg of biotin, 2 ml of trace metals, and deionized waterto 1 liter. The trace metals solution was composed of 1.59 g of ZnSO₄.7H₂O, 0.76 g of CuSO₄.5 H₂O, 7.52 g of FeSO₄.7 H₂O, 1.88 g of MnSO₄.H₂O,20 g of citric acid, and deionized water to 1 liter. Approximately 15 mlof sterile buffer (7.0 g of Na₂HPO₄, 3.0 g of KH₂PO₄, 4.0 g of NaCl, 0.2g of MgSO₄.7 H₂O, and deionized water to 1 liter) was used to gentlywash off some of the cells from the agar surface. The bacterialsuspension was then used for inoculation of baffled shake flaskscontaining 100 ml of growth medium composed of 11 g of soy bean meal,0.4 g of Na₂HPO₄, 5 drops of antifoam, and deionized water to 100 ml.The inoculated shake flasks were incubated at 37° C. for about 20 hourswith shaking at 300 rpm, after which 100 ml (obtained by combining themedia from two independent shake flasks with the same strain) were usedfor inoculation of a 3 liter fermentor with 900 ml of medium composed of40 g of hydrolyzed potato protein, 6 g of K₂SO₄, 4 g of Na₂HPO₄, 12 g ofK₂HPO₄, 4 g of (NH₄)₂SO₄, 0.5 g of CaCO₃, 2 g of citric acid; 4 g ofMgSO₄, 40 ml of trace metals solution (described above), 1 mg of biotin(biotin was added as 1 ml of a 1 g per liter biotin solution in thebuffer described above), 1.3 ml of antifoam, and deionized water to 1liter. The medium was adjusted to pH 5.25 with phosphoric acid prior tobeing autoclaved.

The fermentation was carried out as a fed-batch fermentation withsucrose solution being the feed. The fermentation temperature was heldconstant at 37° C. The tanks were aerated with 3 liter air per minute,and the agitation rate was held in the range of 1,500-1,800 rpm. Thefermentation time was around 60-70 hours. The pH was maintained in therange of pH 6.5-7.3.

The fermentation was assayed for xylanase activity according to thefollowing procedure. Culture supernatants were diluted appropriately in0.1 M sodium acetate pH 5.0. A purified Dictyoglomus thermophilumxylanase was diluted using 2-fold steps starting with a 1.71 μg/mlconcentration and ending with a 0.03 μg/ml concentration in the samplebuffer. A total of 40 μl of each dilution including standard wastransferred to a 96-well flat bottom plate. Using a Biomek NX (BeckmanCoulter, Fullerton Calif., USA), a 96-well pippetting workstation, 40 μlan Azo-Wheat arabinoxylan (Megazyme International, Ireland) substratesolution (1% w/v) was added to each well then incubated at 50° C. for 30minutes. Upon completion of the incubation the reaction was stopped with200 μl of ethanol (95% v/v). The samples were then incubated at ambienttemperatures for 5 minutes followed by centrifugation at 3,000 rpm for10 minutes. One hundred-fifty microliters of the supernatant was removedand dispensed into a new 96-well flat bottom plate. An optical densityof 590 nm was obtained for the 96-well plate using a SPECTRAMAX® 250μlate reader (Molecular Devices, Sunnvale Calif., USA). Sampleconcentrations were determined by extrapolation from the generatedstandard curve.

Filtrated broth was added to 2% v/v GC-850 (Gulbrandsen, S.C., USA)followed by centrifugation at 20,000×g for 20 minutes. The supernatantwas again added 2% v/v GC-850 followed by centrifugation at 20,000×g for20 minutes. The supernatant was concentrated using a tangential flowconcentrator equipped with a Sartocon® Slice cassette with a 10 kDaMW-CO polyethersulfone membrane. The concentrate was 80° C. treated for30 minutes and filtrated through a 1.2 μm glass microfibre filter(Whatman, International Ltd, Maidstone, England). The pH was adjusted topH 8.0 and loaded onto a MEP HyperCel™ (Pall Corporation, East Hills,N.Y. USA) column. The bound proteins were eluted with 50 mM acetic acidpH 4.5. The eluted proteins were stirred with activated carbon 1% w/v(Picatif FGV 120, Pica, France) for 15 minutes and filtrated on 0.2 μmPES filter (Nalge Nunc International, New York, N.Y. USA). The pH wasadjusted to pH 5.0 using 3 M Tris. Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 85: Preparation of Aspergillus aculeatus Strain CBS 172.66 GH3Beta-Xylosidase

The Aspergillus aculeatus strain CBS 172.66 GH beta-xylosidase (SEQ IDNO: 201 [DNA sequence] and SEQ ID NO: 202 [deduced amino acid sequence])was recombinantly prepared according to the following procedure.

Aspergillus aculeatus CBS 172.66 was used as the source of thepolypeptide having beta-xylosidase activity.

Genomic sequence information was generated by the U.S. Department ofEnergy Joint Genome Institute (JGI). A preliminary assembly of thegenome was downloaded from JGI and analyzed using the PedantPro™Sequence Analysis Suite (Biomax Informatics AG, Martinsried, Germany).Gene models constructed by the software were used as a starting pointfor detecting GH3 homologues in the genome. More precise gene modelswere constructed manually using multiple known GH3 protein sequences asa guide.

To generate genomic DNA for PCR amplification, Aspergillus aculeatus CBS172.66 was propagated on PDA agar plates by growing at 26° C. for 7days. Spores harvested from the PDA plates were used to inoculate 25 mlof YP+2% glucose medium in a baffled shake flask and incubated at 26° C.for 48 hours with agitation at 200 rpm.

Genomic DNA was isolated according to a modified FastDNA® SPIN protocol(Qbiogene, Inc., Carlsbad, Calif., USA). Briefly a FastDNA® SPIN Kit forSoil (Qbiogene, Inc., Carlsbad, Calif., USA) was used in a FastPrep® 24Homogenization System (MP Biosciences, Santa Ana, Calif., USA). Two mlof fungal material from the above cultures were harvested bycentrifugation at 14,000×g for 2 minutes. The supernatant was removedand the pellet resuspended in 500 μl of deionized water. The suspensionwas transferred to a Lysing Matrix E FastPrep® tube (Qbiogene, Inc.,Carlsbad, Calif., USA) and 790 μl of sodium phosphate buffer and 100 μlof MT buffer from the FastDNA® SPIN Kit were added to the tube. Thesample was then secured in the FastPrep® Instrument (Qbiogene, Inc.,Carlsbad, Calif., USA) and processed for 60 seconds at a speed of 5.5m/sec. The sample was then centrifuged at 14,000×g for two minutes andthe supernatant transferred to a clean EPPENDORF® tube. A 250 μl volumeof PPS reagent from the FastDNA® SPIN Kit was added and then the samplewas mixed gently by inversion. The sample was again centrifuged at14,000×g for 5 minutes. The supernatant was transferred to a 15 ml tubefollowed by 1 ml of Binding Matrix suspension from the FastDNA® SPIN Kitand then mixed by inversion for two minutes. The sample was placed in astationary tube rack and the silica matrix was allowed to settle for 3minutes. A 500 μl volume of the supernatant was removed and discardedand then the remaining sample was resuspended in the matrix. The samplewas then transferred to a SPIN filter tube from the FastDNA® SPIN Kitand centrifuged at 14,000×g for 1 minute. The catch tube was emptied andthe remaining matrix suspension added to the SPIN filter tube. Thesample was again centrifuged (14,000×g, 1 minute). A 500 μl volume ofSEWS-M solution from the FastDNA® SPIN Kit was added to the SPIN filtertube and the sample was centrifuged at the same speed for 1 minute. Thecatch tube was emptied and the SPIN filter replaced in the catch tube.The unit was centrifuged at 14,000×g for 2 minutes to “dry” the matrixof residual SEWS-M wash solution. The SPIN filter was placed in a freshcatch tube and allowed to air dry for 5 minutes at room temperature. Thematrix was gently resuspended in 100 μl of DES (DNase/Pyrogen freewater) with a pipette tip. The unit was centrifuged (14,000×g, 1 minute)to elute the genomic DNA followed by elution with 100 μl of 10 mM Tris,0.1 mM EDTA, pH 8.0 by renewed centrifugation at 14,000×g for 1 minuteand the eluates were combined. The concentration of the DNA harvestedfrom the catch tube was measured by a UV spectrophotometer at 260 nm.

Synthetic oligonucleotide primers shown below are designed to PCRamplify Aspergillus aculeatus CBS 172.66 GH3 genes from the genomic DNAprepared in Example 2. An IN-FUSION™ Cloning Kit (Clontech, MountainView, Calif., USA) is used to clone the fragments directly into theexpression vector pDau109 (WO 2005/042735).

Primer GH3-114f: (SEQ ID NO: 298)5′-ACACAACTGGGGATCCACCATGGCTGTGGCGGCTCTT-3′ Primer GH3-114r:(SEQ ID NO: 299) 5′-AGATCTCGAGAAGCTTACTACTCATCCCCCTGCAC-3′

PCR reactions are carried out with genomic DNA prepared from Example 2for amplification of the genes identified in Example 1. The PCR reactionis composed of 1 μl of genomic DNA, 1 μl of primer forward (f) (50 μM);1 μl of primer reverse (r) (50 μM); 10 μl of 5×HF buffer, 2 μl of 10 mMdNTP; 1 μl of PHUSION® DNA polymerase, and PCR-grade water up to 50 μl.Primers GH3-114f and GH3-114r are used simultaneously.

The PCR reactions are performed using a DYAD® PCR machine programmed for2 minutes at 98° C. followed by 20 touchdown cycles at 98° C. for 15seconds, 70° C. (−1° C./cycle) for 30 seconds, and 72° C. for 2 minutes30 seconds; and 25 cycles each at 98° C. for 15 seconds, 60° C. for 30seconds, 72° C. for 2 minutes 30 seconds; and 5 minutes at 72° C.

The reaction products are isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE)buffer where approximately 2.5 to 3.0 kb PCR product bands are excisedfrom the gels and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the A. aculeatus GH3 genes are cloned into theexpression vector pDAu109 (WO 2005042735) linearized with Barn HI andHind III, using an IN-FUSION™ Dry-Down PCR Cloning Kit according to themanufacturer's instructions.

A 2.5 μl volume of the five times diluted ligation mixture is used totransform E. coli TOP10 chemically competent cells. Five colonies areselected on LB agar plates containing 100 μg of ampicillin per ml andcultivated overnight in 3 ml of LB medium supplemented with 100 μg ofampicillin per ml. Plasmid DNA is purified using an E.Z.N.A.® PlasmidMini Kit according to the manufacturer's instructions. The Aspergillusaculeatus GH3 gene sequences were verified by Sanger sequencing with anApplied Biosystems Model 3700 Automated DNA Sequencer using version 3.1BIG-DYE™ terminator chemistry (Applied Biosystems, Inc., Foster City,Calif., USA) (Applied Biosystems, Inc., Foster City, Calif., USA).Nucleotide sequence data are scrutinized for quality and all sequencesare compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

The coding sequence is 2412 bp including the stop codon and contains nointrons. The encoded predicted protein is 803 amino acids. Using theSignalP program (Nielsen et al., 1997, supra), a signal peptide of 17residues was predicted. The predicted mature protein contains 786 aminoacids.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice in total on COVE sucrose medium (Cove, 1996, Biochim.Biophys. Acta 133: 51-56) containing 1 M sucrose and 10 mM sodiumnitrate, supplemented with 10 mM acetamide and 15 mM CsCl. Fermentationwas then carried out in 250 ml shake flasks using DAP-2C-1 medium for 4days at 30° C. with shaking at 100 rpm. The fermentation broth wasfiltered using standard methods.

Ammonium sulphate was added to the filtrated broth to a consentration of2 M. After filtration using a 0.2 μm PES filter (Thermo FisherScientific, Roskilde, Denmark), the filtrate was loaded onto a PhenylSepharose™ 6 Fast Flow column (high sub) (GE Healthcare, Piscataway,N.J., USA) equilibrated in 25 mM HEPES pH 7.0 with 2 M ammoniumsulphate, and bound proteins were eluted with 25 mM HEPES pH 7.0 with noammonium sulphate. The fractions were pooled and applied to a Sephadex™G-25 (medium) (GE Healthcare, Piscataway, N.J., USA) column equilibratedin 25 mM HEPES pH 7.0. The fractions were pooled and then applied to aSOURCE™ 15Q (GE Healthcare, Piscataway, N.J., USA) column equilibratedin 25 mM HEPES pH 7.0, and bound proteins were eluted with a lineargradient from 0-1000 mM sodium chloride. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 86: Preparation of Aspergillus aculeatus Strain CBS 186.67 GH3Beta-Xylosidase

The Aspergillus aculeatus strain CBS 186.67 GH3 beta-xylosidase (SEQ IDNO: 203 [DNA sequence] and SEQ ID NO: 204 [deduced amino acid sequence])was recombinantly prepared according to the following procedure.

Genomic DNA was isolated according to the procedure described in Example73.

The Aspergillus aculeatus beta-xylosidase gene was isolated by PCR usingtwo cloning primers GH3-101f and GH3-101r, shown below, which weredesigned based on the publicly available Aspergillus aculeatus xyl2full-length sequence (GenBank AB462375.1) for direct cloning using theIN-FUSION™ strategy.

Primer GH3-101f: (SEQ ID NO: 300)5′-acacaactggggatccaccatggctgtggcggctcttgctctgc tgg-3′ Primer GH3-101r:(SEQ ID NO: 301) 5′-agatctcgagaagcttaCTCATCCCCCGCCACCCCCTGCACCTC C-3′

A PCR reaction was performed with genomic DNA prepared from Aspergillusaculeatus CBS 186.67 in order to amplify the full-length gene. The PCRreaction was composed of 1 μl of genomic DNA, 0.75 μl of primerGH3-101.1f (10 μM), 0.75 μl of primer GH3-101.1r (10 μM), 3 μl of 5×HFbuffer, 0.25 μl of 50 mM MgCl₂, 0.3 μl of 10 mM dNTP, 0.15 μl ofPHUSION® DNA polymerase, and PCR-grade water up to 15 μl. The PCRreaction was performed using a DYAD® PCR machine programmed for 2minutes at 98° C. followed by 10 touchdown cycles at 98° C. for 15seconds, 70° C. (−1° C./cycle) for 30 seconds, and 72° C. for 2 minutes30 seconds; and 25 cycles each at 98° C. for 15 seconds, 60° C. for 30seconds, 72° C. for 2 minutes 30 seconds, and 5 minutes at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where an approximately 2.4 kb PCR product band wasexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit according to manufacturer's instructions. DNAcorresponding to the Aapergillus aculeatus beta-xylosidase gene wascloned into the expression vector pDAu109 (WO 2005042735) linearizedwith Barn HI and Hind III, using an IN-FUSION™ Dry-Down PCR Cloning Kitaccording to the manufacturer's instructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells. Three colonies were selected onLB agar plates containing 100 μg of ampicillin per ml and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using an E.Z.N.A.® Plasmid Mini Kitaccording to the manufacturer's instructions. The Aspergillus aculeatusbeta-xylosidase gene sequence was verified by Sanger sequencing beforeheterologous expression.

The coding sequence is 2454 bp including the stop codon. The gene doesnot contain introns. The encoded predicted protein is 817 amino acids.Using the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 17 residues was predicted. The predictedmature protein contains 800 amino acids.

Protoplasts of Aspergillus oryzae MT3568 were prepared as described inWO 95/02043. A. oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene with the pyrG gene. One hundred microliters of protoplastsuspension were mixed with 2.5-15 μg of the Aspergillus expressionvector and 250 μl of 60% PEG 4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH7.5 were added and gently mixed. The mixture was incubated at 37° C. for30 minutes and the protoplasts were spread on COVE sucrose (1 M) platessupplemented with 10 mM acetamide and 15 mM CsCl for transformantselection. After incubation for 4-7 days at 37° C. spores of severaltransformants were seeded on YP-2% maltodextrin medium. After 4 dayscultivation at 30° C. culture broth was analyzed in order to identifythe best transformants based on their ability to produce a large amountof active Aculeatus aculeatus beta-xylosidase. The screening was basedon intensity of the band corresponding to the heterologous expressedprotein determined by SDS-PAGE and activity of the enzyme on4-nitrophenyl-beta-D-xylopyranoside (pNPX) as follows. Ten μl of culturebroth was mixed with 90 μl of assay reagent containing 10 μl of 0.1%TWEEN® 20, 10 μl of 1 M sodium citrate pH 5, 4 μl of 100 mM of pNPXsubstrate (Sigma Aldrich) solubilized in DMSO (0.4% final volume instock solution), and filtered water. The assay was performed for 30minutes at 37° C. and absorbance determined at 405 nm before and afteraddition of 100 μl of 1 M sodium carbonate pH 10. The highest absorbancevalues at 405 nm were correlated to the SDS-PAGE data for selection ofthe best transformant.

Spores of the best transformant designated A. oryzae EXP3611 were spreadon COVE plates containing 0.01% TRITON® X-100 in order to isolate singlecolonies. The spreading was repeated twice in total on COVE sucrosemedium (Cove, 1996, Biochim. Biophys. Acta 133: 51-56) containing 1 Msucrose and 10 mM sodium nitrate, supplemented with 10 mM acetamide and15 mM CsCl. Fermentation was then carried out in 250 ml shake flasksusing DAP-4C-1 medium for 4 days at 30° C. with shaking at 100 rpm. Thefermentation broth was filtered using standard methods. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 87: Preparation of Aspergillus fumigatus Strain NN051616 GH3Beta-Xylosidase Q0H905

The Aspergillus fumigatus strain NN051616 GH3 beta-xylosidase (SEQ IDNO: 205 [DNA sequence] and SEQ ID NO: 206 [deduced amino acid sequence])was recombinantly prepared according to the following procedure.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Aspergillus fumigatus beta xylosidase gene from the genomicDNA. An InFusion Cloning Kit (Clontech, Mountain View, Calif.) was usedto clone the fragment directly into the expression vector, pAILo2 (WO2005/074647), without the need for restriction digests and ligation.

Forward primer: (SEQ ID NO: 302)5′-ACTGGATTTACCATGGCGGTTGCCAAATCTATTGCT-3′ Reverse primer:(SEQ ID NO: 303) 5′-TCACCTCTAGTTAATTAATCACGCAGACGAAATCTGCT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAILo2.

Fifteen picomoles of each of the primers above were used in a PCRreaction containing 250 ng of Aspergillus fumigatus genomic DNA, 1×Expand High Fidelity Buffer with MgCl₂ (Roche Applied Science,Indianapolis, Ind.), 1 μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP,0.75 units of Expand High fidelity Enzyme Mix (Roche Applied Science,Indianapolis, Ind.), in a final volume of 50 μl. The amplificationconditions were one cycle at 94° C. for 2 minutes; 10 cycles each at 94°C. for 15 seconds, 56.5° C. for 30 seconds, and 72° C. for 2 minutes;and 20 cycles each at 94° C. for 15 seconds, 56.5° C. for 30 seconds,and 72° C. for 2 minutes plus 5 seconds per successive cycle. The heatblock was then held at 72° C. for 7 minutes followed by a 4° C. soakcycle.

The reaction products were isolated on a 1.0% agarose gel using TAEbuffer and a 2.4 kb product band was excised from the gel and purifiedusing a MinElute® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.)according to the manufacturer's instructions.

The fragment was then cloned into pAILo2 using an InFusion Cloning Kit.The vector was digested with Nco I and Pac I (using conditions specifiedby the manufacturer). The fragment was purified by gel electrophoresisand QlAquick kit (QIAGEN Inc., Valencia, Calif.) gel purification. Thegene fragment and the digested vector were combined together in areaction resulting in the expression plasmid pAG57, in whichtranscription of the Aspergillus fumigatus beta-xylosidase gene wasunder the control of the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase). The recombinationreaction (20 μl) was composed of 1× InFusion Buffer (Clontech, MountainView, Calif.), 1×BSA (Clontech, Mountain View, Calif.), 1 μl of InFusionenzyme (diluted 1:10) (Clontech, Mountain View, Calif.), 182 ng ofpAILo2 digested with Nco I and Pac I, and 97.7 ng of the Aspergillusfumigatus beta-xylosidase purified PCR product. The reaction wasincubated at 37° C. for 15 minutes followed by 15 minutes at 50° C. Thereaction was diluted with 40 μl of TE buffer and 2.5 μl of the dilutedreaction was used to transform E. coli Top10 Competent cells. An E. colitransformant containing pAG57 (Aspergillus fumigatus beta-xylosidasegene) was identified by restriction enzyme digestion and plasmid DNA wasprepared using a BIOROBOT® 9600. The pAG57 plasmid construct wassequenced using an Applied Biosystems 3130x1 Genetic Analyzer (AppliedBiosystems, Foster City, Calif., USA) to verify the sequence.

Aspergillus oryzae JaL355 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422 andtansformed with 5 μg of pAG57. Twenty-four transformants were isolatedto individual PDA plates.

Plugs taken from the original transformation plate of each of thetwenty-four transformants were added to 1 ml of M410 separately in 24well plates, which were incubated at 34° C. After three days ofincubation, 7.5 μl of supernatant from each culture was analyzed usingCriterion stain-free, 8-16% gradient SDS-PAGE, (BioRad, Hercules,Calif.) according to the manufacturer's instructions. SDS-PAGE profilesof the cultures showed that several transformants had a new major bandof approximately 130 kDa.

Confluent PDA plate of the highest expressing transformant was washedwith 5 ml of 0.01% TWEEN® 20 and inoculated into a 500 ml Erlenmeyerflask containing 100 ml of M410 medium. Inoculated flask was incubatedwith shaking for 3 days at 34° C. The broth was filtered through a 0.22μm stericup suction filter (Millipore, Bedford, Mass.).

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator (Pall Filtron, Northborough, Mass., USA) equipped witha 10 kDa polyethersulfone membrane (Pall Filtron, Northborough, Mass.,USA) with 50 mM sodium acetate pH 5.0. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 88: Evaluation of Two Cellobiohydrolases I Replacing a CBHIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of two cellobiohydrolase I proteins to replace a CBHIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 37% CBHI, 25% Aspergillus fumigatus Cel6A CBHII,10% Myceliophthora thermophila Cel5A EGII, 15% Penicillium sp. GH61Apolypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus Cel3A beta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3xylanase, and 3% Trichoderma reesei GH3 beta-xylosidase.

The following CBHIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel7A CBHI and Penicillium emersoniiCel7A CBHI.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 29, Penicillium emersonii Cel7A CBHI performed the sameas Aspergillus fumigatus Cel7A CBHI at 50° C. and performed better thanAspergillus fumigatus Cel7A CBHI at 55-65° C. (as the degree ofcellulose conversion to glucose was higher for Penicillium emersoniiCel7A CBHI than Aspergillus fumigatus Cel7A CBHI at 55-65° C.).

Example 89: Evaluation of Two Cellobiohydrolases I Replacing a CBHIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of two cellobiohydrolase I proteins to replace a CBHIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 40% CBHI, 25% Aspergillus fumigatus Cel6A CBHII,10% Myceliophthora thermophila Cel5A EGII, 15% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus GH10 xylanase, and 5% Aspergillus fumigatus Cel3Abeta-glucosidase.

The following CBHIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel7A CBHI and Penicillium pinophilumCel7A CBHI. The high-temperature composition including Aspergillusfumigatus Cel7A CBHI was loaded at 3.3 mg total protein per gramcellulose instead of 3 mg total protein per gram cellulose, in whichPenicillium pinophilum Cel7A CBHI was loaded.

The assay was performed as described in Example 34. The 1 ml reactionswith 5% milled unwashed PCS were conducted for 72 hours in 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

As shown in FIG. 30, Penicillium pinophilum Cel7A CBHI performed well inthe entire range of temperatures. Penicillium pinophilum Cel7A CBHIperformed about the same as Aspergillus fumigatus Cel7A CBHI at 50° C.and 55° C., but the performance was slightly lower compared toAspergillus fumigatus Cel7A CBHI at 60° C. and 65° C.

Example 90: Evaluation of Two Cellobiohydrolases I Replacing a CBHIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of two cellobiohydrolase I proteins to replace a CBHIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 37% CBHI, 25% Aspergillus fumigatus Cel6A CBHII,10% Myceliophthora thermophila Cel5A EGII, 15% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus Cel3A beta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3xylanase, and 3% Trichoderma reesei GH3 beta-xylosidase.

The following CBHIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel7A CBHI and Aspergillus terreusCel7A CBHI.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 31, the performance of Aspergillus terreus Cel7A CBHIwas the same as the performance of Aspergillus fumigatus Cel7A CBHI at55° C. and lower at 60-65° C.; however, at 50° C., the degree ofcellulose conversion to glucose was much higher for Aspergillus terreusCel7A CBHI than Aspergillus fumigatus Cel7A CBHI at 50° C.

Example 91: Evaluation of Three Cellobiohydrolases I Replacing a CBHIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-60° C.

The ability of three cellobiohydrolase I proteins to replace a CBHIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., and 60° C. using milledunwashed PCS as a substrate. The high-temperature enzyme compositionincluded 45% CBHI, 25% Thielavia terrestris Cel6A CBHII, 5% Trichodermareesei Cel7B EGI, 5% Thermoascus aurantiacus Cel5A EGII, 5% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Thielavia terrestris GH61E polypeptide having cellulolytic enhancingactivity, 5% Aspergillus fumigatus Cel3A beta-glucosidase, and 5%Aspergillus fumigatus GH10 xyn3 xylanase.

The following CBHIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel7A CBHI, Neosartorya fischeriCel7A CBHI, and Aspergillus nidulans Cel7A CBHI.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 32, the performance of Neosartorya fischeri Cel7A CBHIwas lower than the performance of Aspergillus fumigatus Cel7A CBHI at50° C. and 55° C., but performance was the same at 60° C. Aspergillusnidulans Cel7A CBHI performed almost the same as Aspergillus fumigatusCel7A CBHI at 50° C., but showed lower hydrolysis at 55° C. and 60° C.

Example 92: Evaluation of Two Cellobiohydrolases II Replacing a CBHIIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of two cellobiohydrolase II proteins to replace a CBHIIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 37% Aspergillus fumigatus Cel7A CBHI, 25% CBHII,10% Myceliophthora thermophila Cel5A EGII, 15% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus Cel3A beta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3xylanase, and 3% Trichoderma reesei GH3 beta-xylosidase.

The following CBHIIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel6A CBHII and Finnellia nivea Cel6ACBHII.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 33, Finnellia nivea Cel6A CBHII performed well at50-55° C. as it showed hydrolysis levels similar to Aspergillusfumigatus Cel6A CBHII at 50-55° C., but the performance declined at60-65° C.

Example 93: Evaluation of Three Cellobiohydrolases II Replacing a CBHIIComponent in a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-65° C.

The ability of three cellobiohydrolase II proteins to replace a CBHIIcomponent in a high-temperature enzyme composition (3 mg total proteinper g cellulose) was tested at 50° C., 55° C., 60° C., and 65° C. usingmilled unwashed PCS as a substrate. The high-temperature enzymecomposition included 37% Aspergillus fumigatus Cel7A CBHI, 25% CBHII,10% Trichoderma reesei Cel5A EGII, 15% Penicillium sp. GH61A polypeptidehaving cellulolytic enhancing activity, 5% Aspergillus fumigatus Cel3Abeta-glucosidase, 5% Aspergillus fumigatus GH10 xyn3 xylanase, and 3%Trichoderma reesei GH3 beta-xylosidase.

The following CBHIIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel6A CBHII, Penicillium emersoniiCel6A CBHII, and Penicillium pinophilum Cel6A CBHII.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 34, Penicillium emersonii Cel6A CBHII performed well inthe entire range of temperatures. Penicillium emersonii Cel6A CBHIIperformed almost as well as Aspergillus fumigatus Cel6A CBHII at 50° C.,but performed slightly lower than Aspergillus fumigatus Cel6A CBHII at55-65° C. The performance of Penicillium pinophilum Cel6A CBHII wascomparable to that of Aspergillus fumigatus Cel6A CBHII at 50° C. and55° C.; however, performance declined at 60° C. and 65° C.

Example 94: Evaluation of Three Endoglucanases II Replacing anEndoglucanase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

The ability of three endoglucanase II proteins to replace anendoglucanase component in a high-temperature enzyme composition (3 mgtotal protein per g cellulose) was tested at 50° C., 55° C., 60° C., and65° C. using milled unwashed PCS as a substrate. The high-temperatureenzyme composition included 40% Aspergillus fumigatus Cel7A CBHI, 25%Aspergillus fumigatus Cel6A CBHII, 10% EG cellulase, 15% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Aspergillus fumigatus Cel3A beta-glucosidase, and 5% Aspergillusfumigatus GH10 xyn3 xylanase.

The following EGIIs were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus Cel5A EGII, Neosartorya fischeriCel5A EGII, and Myceliophthora thermophila Cel5A EGII.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 35, all endoglucanase II proteins performed similarlywithin this temperature range, with Neosartorya fischeri Cel5A EGII andMyceliophthora thermophila Cel5A EGII having similar activity at 50° C.and 55° C. and Aspergillus fumigatus Cel5A EGII having comparableactivity to Neosartorya fischeri Cel5A EGII and Myceliophthorathermophila Cel5A EGII at 60° C. and 65° C.

Example 95: Evaluation of Two Beta-Glucosidases Replacing aBeta-Glucosidase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

The ability of three beta-glucosidase proteins to replace abeta-glucosidase component in a high-temperature enzyme composition (3mg total protein per g cellulose) was tested at 50° C., 55° C., 60° C.,and 65° C. using milled unwashed PCS as a substrate. Thehigh-temperature enzyme composition included 37% Aspergillus fumigatusCel7A CBHI, 25% Aspergillus fumigatus Cel6A CBHII, 10% Myceliophthorathermophila Cel5A EGII, 15% Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity, 5% beta-glucosidase, 5%Aspergillus fumigatus GH10 xyn3 xylanase, and 3% Trichoderma reesei GH3beta-xylosidase.

The following beta-glucosidases were each tested in the high-temperatureenzyme composition: Aspergillus fumigatus Cel3A beta-glucosidase andAspergillus aculeatus beta-glucosidases.

The assay was performed as described in Example 34, with the exceptionof glucose background, in which 40 g per liter of glucose was includedin the reactions. The 1 ml reactions with milled unwashed PCS (5%insoluble solids) were conducted for 72 hours in 50 mM sodium acetate pH5.0 buffer containing 1 mM manganese sulfate. All reactions wereperformed in triplicate and involved single mixing at the beginning ofhydrolysis.

As shown in FIG. 36, Aspergillus aculeatus Cel3A beta-glucosidase hadslightly higher or similar performance as Aspergillus fumigatus Cel3Abeta-glucosidase at all temperatures

Example 96: Evaluation of Four Beta-Glucosidases Replacing aBeta-Glucosidase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-60° C.

Four beta-glucosidases, including Aspergillus fumigatus Cel3Abeta-glucosidase, Aspergillus kawashii Cel3A beta-glucosidase,Aspergillus clavatus Cel3 beta-glucosidase, and Talaromyces emersoniiCel3A beta-glucosidase were each evaluated in a high-temperature enzymecomposition at 50° C., 55° C., and 60° C. using milled unwashed PCS as asubstrate. The high-temperature enzyme composition included 45%Aspergillus fumigatus Cel7A CBHI, 25% Thielavia terrestris Cel6A CBHII,5% Trichoderma reesei Cel7B EGI, 5% Thermoascus aurantiacus Cel5A EGII,5% Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity, 5% Thielavia terrestris GH61E polypeptide havingcellulolytic enhancing activity, 5% Aspergillus fumigatus GH10 xyn3xylanase, and 5% beta-glucosidase. The high-temperature enzymecomposition was used at 3.0 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 37 demonstrated that all beta-glucosidasesexcept Talaromyces emersonii Cel3A beta-glucosidase had similarperformance all three temperatures. Talaromyces emersonii Cel3Abeta-glucosidase had lower activity at 50° C. and 55° C. but hadequivalent activity at 60° C.

Example 97: Evaluation of Three Beta-Glucosidases Replacing aBeta-Glucosidase Component in a High-Temperature Enzyme Composition inSaccharification of Milled Unwashed PCS at 50-65° C.

Three beta-glucosidases, including Aspergillus fumigatus Cel3Abeta-glucosidase, Penicillium oxalicum Cel3A beta-glucosidase (Example77), and Penicillium oxalicum Cel3A beta-glucosidase (Example 78) wereeach evaluated in a high-temperature enzyme composition at 50° C., 55°C., 60° C., and 65° C. using milled unwashed PCS as a substrate. Thehigh-temperature enzyme composition included 40% Aspergillus fumigatusCel7A CBHI, 25% Aspergillus fumigatus Cel6A CBHII, 10% Myceliophthorathermophila Cel5A EGII, 15% Thermoascus aurantiacus GH61A polypeptidehaving cellulolytic enhancing activity, 5% Aspergillus fumigatus GH10xyn3 xylanase, and 5% beta-glucosidase. The high-temperature enzymecomposition was used at 3.0 mg total protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 38 demonstrated that the beta-glucosidases hadsimilar activity at all temperatures.

Example 98: Evaluation of the Ability of Three GH61 Polypeptides HavingCellulolytic Enhancing Activity to Enhance PCS-Hydrolyzing Activity of aHigh-Temperature Enzyme Composition at 50-65° C. Using Milled Washed PCS

The ability of three GH61 polypeptides having cellulolytic enhancingactivity, Thermoascus aurantiacus GH61A, Penicllium sp GH61A, andThermoascus crustaceus GH61A, were each evaluated for their ability toenhance the PCS-hydrolyzing activity of a high-temperature enzymecomposition using milled washed PCS at 50° C., 55° C., 60° C., and 65°C. Each GH61 polypeptide was separately added at 11.6% enzyme to a hightemperature enzyme mixture. The high-temperature enzyme compositionincluded 43.5% Aspergillus fumigatus Cel7A CBHI, 29% Aspergillusfumigatus Cel6A CBHII, 12% Myceliophthora thermophila Cel5A EGII, 6%Aspergillus fumigatus Cel3A beta-glucosidase, 6% Aspergillus fumigatusGH10 xyn3 xylanase, and 4% Trichoderma reesei GH3 beta-xylosidase. Theresults for the enzyme compositions containing GH61 polypeptides (2.3725mg total protein per g cellulose) were compared with the results for asimilar enzyme composition to which no GH61 polypeptide was added (2.125mg total protein per g cellulose).

The assay was performed as described in Example 34. The 1 ml reactionswith milled washed PCS (5% insoluble solids) were conducted for 72 hoursin 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.All reactions were performed in triplicate and involved single mixing atthe beginning of hydrolysis.

As shown in FIG. 39, all three GH61 polypeptides showed significantcellulase-enhancing activity, with Thermoascus aurantiacus GH61Apolypeptide, Penicllium sp GH61A polypeptide, and Thermoascus crustaceusGH61A polypeptide having similar enhancement at 50° C. and 55° C. whileThermoascus aurantiacus GH61A polypeptide had higher activity thanPenicllium sp GH61A polypeptide and Thermoascus crustaceus GH61Apolypeptide at 60° C. and 65° C.

Example 99: Evaluation of Three Xylanases Replacing a Xylanase Componentin a High-Temperature Enzyme Composition in Saccharification of MilledUnwashed PCS at 50-65° C.

The ability of three xylanases to replace an xylanase component in ahigh-temperature enzyme composition (3 mg total protein per g cellulose)was tested at 50° C., 55° C., 60° C., and 65° C. using milled unwashedPCS as a substrate. The high-temperature enzyme composition included 37%Aspergillus fumigatus Cel7A CBHI, 25% Aspergillus fumigatus Cel6A CBHII,10% Myceliophthora thermophila Cel5A EGII, 15% Thermoascus aurantiacusGH61A polypeptide having cellulolytic enhancing activity, 5% Aspergillusfumigatus Cel3A beta-glucosidase, 3% Trichoderma reesei GH3beta-xylosidase and 5% xylanase.

The following xylanases were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus GH10 xylanase 3, Talaromycesemersonii GH10 xylanase, and Penicillium emersonii GH10 xylanase.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 40, Talaromyces emersonii GH10 xylanase and Penicilliumemersonii GH10 xylanase had similar activity as Aspergillus fumigatusGH10 xylanase 3 at all three temperatures.

Example 100: Evaluation of Three Xylanases by Adding a XylanaseComponent to a High-Temperature Enzyme Composition in Saccharificationof Milled Unwashed PCS at 50-60° C.

Three xylanases were each evaluated as a 10% addition to ahigh-temperature enzyme composition (3.5 mg total protein per gcellulose) at 50° C., 55° C., and 60° C. using milled unwashed PCS as asubstrate. The high-temperature enzyme composition included 45%Aspergillus fumigatus Cel7A CBHI, 25% Myceliophthora thermophila Cel6ACBHII, 10% Myceliophthora thermophila Cel5A EGII, 5% Thermoascusaurantiacus GH61A polypeptide having cellulolytic enhancing activity, 5%Thielavia terrestris GH61E polypeptide having cellulolytic enhancingactivity, and 5% Penicillium brasilianum Cel3A beta-glucosidase.

The following xylanases were each tested in the high-temperature enzymecomposition: Aspergillus fumigatus GH10 xylanase (xyl3), Meripilusgiganteus GH10 xylanase, and Dictyoglomus thermophilum GH11 xylanase.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 41, all three xylanase increased hydrolysis of thehigh-temperature enzyme composition. Aspergillus fumigatus GH10 xylanaseand Meripilus giganteus GH10 xylanase had the same activity at 50° C.and 55° C. but Aspergillus fumigatus GH10 xylanase 3 had significantlyhigher activity at 60° C. than Meripilus giganteus GH10 xylanase.Dictyoglomus thermophilum GH11 xylanase had lower activity thanAspergillus fumigatus GH10 xylanase 3 at all three temperatures butDictyoglomus thermophilum GH11 xylanase had increasing activity astemperature increases to 60° C.

Example 101: Comparison of XCL-602-Based Enzyme Compositions ContainingDifferent Cellobiohydrolases and Xylanases in Hydrolysis of MilledUnwashed PCS at 50-60° C.

Four XCL-602 based enzyme compositions containing a differentcellobiohydrolase and xylanase were tested at 50° C., 55° C., and 60° C.using milled unwashed PCS as a substrate. The cellobiohydrolases testedin the XCL-602 based enzyme compositions were Aspergillus fumigatusCel7A CBHI and Penicillium emersonii Cel7 CBHI. The xylanases testedwere Aspergillus fumigatus GH10 xylanase 3 and Trichophaea saccata GH10xylanase. The XCL-602 based enzyme compositions included 40% XCL-602,20% CBHI, 20% Aspergillus fumigatus Cel6A CBHII, 12.5% Penicilliumspecies GH61A, 5% xylanase, and 2.5% Talaromyces emersonii GH3beta-xylosidase. XCL-602 based enzyme compositions containingAspergillus fumigatus Cel7A CBHI were tested at 3.0, 5.0, and 7.0 mgprotein per g cellulose while XCL-602 based enzyme compositionscontaining Penicillium emersonii Cel7 CBHI were tested at 3.0 mg proteinper g cellulose. For comparison, Trichoderma reesei-based XCL-602cellulase was tested at 3.0, 5.0, and 7.0 mg protein per g cellulose.

The assay was performed as described in Example 34. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results for 3 mg protein per g cellulose are shown in FIG. 42. Thetwo xylanases, Aspergillus fumigatus GH10 xylanase 3 and Trichophaeasaccata GH10 xylanase showed similar performance in the XCL-602 basedenzyme compositions with either Aspergillus fumigatus Cel7A CBHI orPenicillium emersonii Cel7 CBHI. For the CBHIs in the XCL-602 basedenzyme compositions with either xylanase, Penicillium emersonii Cel7CBHI had slightly higher performance than Aspergillus fumigatus Cel7ACBHI at all three temperatures. Finally, all XCL-602 based enzymecompositions replaced with CBHI, CBHII, GH61, xylanase, andbeta-xylosidase had significantly higher hydrolysis over thenon-replaced Trichoderma reesei-based XCL-602 cellulase at all threetemperatures. At 80% glucose conversion, the XCL-602 based enzymecompositions containing Aspergillus fumigatus Cel7A CBHI and eitherxylanase at 55° C. required 4.3 mg protein per g cellulose while XCL-602at 50° C. required 6.5 mg protein per g cellulose, a 1.5-fold reductionin protein loading.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-24. (canceled)
 25. An isolated recombinant host cell encoding anenzyme composition comprising: (I) a polypeptide havingcellobiohydrolase I activity selected from the group consisting of: (a)a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 6; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 5; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO: 6; (II) apolypeptide having cellobiohydrolase II activity selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 17, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 17, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 18; (III) a polypeptide having beta-glucosidase activity selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 28; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 27, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 27, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 27; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 28; and (IV) a Family 10 polypeptide having xylanase activity isselected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 48; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 47, (ii)the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 47, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 47; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 48; (V) a polypeptide having beta-xylosidaseactivity selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the mature polypeptide of SEQ ID NO: 60; (b) a polypeptide encoded bya polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:59, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 59, or (iii) the full-length complement of (i) or(ii), wherein high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 59; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 60; wherein saccharification of a cellulosicmaterial is performed with the enzyme composition at a temperature inthe range of about 40° C. to about 70° C.
 26. The isolated recombinanthost cell of claim 25, wherein the enzyme composition further comprisesa GH61 polypeptide having cellulolytic enhancing activity.
 27. Theisolated recombinant host cell of claim 26, wherein the GH61 polypeptidehaving cellulolytic enhancing activity is one or more polypeptidesselected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 34 or 42; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 33 or 41,(ii) the cDNA sequence of the mature polypeptide coding sequence of SEQID NO: 33 or 41, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 33 or 41; and (d) a polypeptide comprising themature polypeptide of SEQ ID NO: 34 or
 42. 28. The isolated recombinanthost cell of claim 25, wherein the enzyme composition further comprisesa Family 11 polypeptide having xylanase activity.
 29. The isolatedrecombinant host cell of claim 28, wherein the xylanase is selected fromthe group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 200; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 199 or its full-lengthcomplement, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamideand washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65°C.; (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 199; and (d) a polypeptidecomprising the mature polypeptide of SEQ ID NO:
 200. 30. The isolatedrecombinant host cell of claim 25, wherein the enzyme compositionfurther comprises one or more polypeptides having endoglucanaseactivity.
 31. The isolated recombinant host cell of claim 30, whereinthe polypeptide has endoglucanase I activity and is selected from thegroup consisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 20; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 19, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 19, or(iii) the full-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19; and (d) a polypeptide comprising the mature polypeptide of SEQID NO:
 20. 32. The isolated recombinant host cell of claim 30, whereinthe polypeptide has endoglucanase II activity and is selected from thegroup consisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 26; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 25, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 25, or(iii) the full-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 25; and (d) a polypeptide comprising the mature polypeptide of SEQID NO:
 26. 33. A method of producing an enzyme composition, comprising:(a) cultivating the recombinant host cell of claim 25 under conditionsconducive for production of the enzyme composition; and (b) recoveringthe enzyme composition.
 34. A method for degrading or converting acellulosic material, comprising: treating the cellulosic material withan enzyme composition comprising: (I) a polypeptide havingcellobiohydrolase I activity selected from the group consisting of: (a)a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 6; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 5; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO: 6; (II) apolypeptide having cellobiohydrolase II activity selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 17, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 17, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 18; (III) a polypeptide having beta-glucosidase activity selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 28; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 27, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 27, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 27; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 28; and (IV) a Family 10 polypeptide having xylanase activity isselected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 48; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 47, (ii)the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 47, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 47; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 48; (V) a polypeptide having beta-xylosidaseactivity selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the mature polypeptide of SEQ ID NO: 60; (b) a polypeptide encoded bya polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:59, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 59, or (iii) the full-length complement of (i) or(ii), wherein high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 59; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 60; wherein the degrading or converting of thecellulosic material is performed with the enzyme composition at atemperature in the range of about 40° C. to about 70° C.
 35. The methodof claim 34, wherein the enzyme composition further comprises a GH61polypeptide having cellulolytic enhancing activity.
 36. The method ofclaim 35, wherein the GH61 polypeptide having cellulolytic enhancingactivity is one or more polypeptides selected from the group consistingof: (a) a polypeptide comprising an amino acid sequence having at least95% sequence identity to the mature polypeptide of SEQ ID NO: 34 or 42;(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 33 or 41, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 33 or 41, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 33 or 41; and (d) a polypeptide comprising the mature polypeptide ofSEQ ID NO: 34 or
 42. 37. The method of claim 34, wherein the enzymecomposition further comprises a Family 11 polypeptide having xylanaseactivity.
 38. The method of claim 37, wherein the xylanase is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 200; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 199 or its full-lengthcomplement, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamideand washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65°C.; (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 199; and (d) a polypeptidecomprising the mature polypeptide of SEQ ID NO:
 200. 39. The method ofclaim 34, wherein the enzyme composition further comprises one or morepolypeptides having endoglucanase activity.
 40. The method of claim 39,wherein the polypeptide has endoglucanase I activity and is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 20; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 19, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 19, or(iii) the full-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19; and (d) a polypeptide comprising the mature polypeptide of SEQID NO:
 20. 41. The method of claim 39, wherein the polypeptide hasendoglucanase II activity and is selected from the group consisting of:(a) a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 26; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 25, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 25, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 25; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO:
 26. 42. Themethod of claim 34, wherein saccharification of the cellulosic materialis performed with the enzyme composition at a temperature in the rangeof about 50° C. to about 65° C.
 43. A method for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising: (I) a polypeptide havingcellobiohydrolase I activity selected from the group consisting of: (a)a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 6; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 5; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO: 6; (II) apolypeptide having cellobiohydrolase II activity selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 17, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 17, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 18; (III) a polypeptide having beta-glucosidase activity selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 28; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 27, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 27, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 27; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 28; and (IV) a Family 10 polypeptide having xylanase activity isselected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 48; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 47, (ii)the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 47, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 47; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 48; (V) a polypeptide having beta-xylosidaseactivity selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the mature polypeptide of SEQ ID NO: 60; (b) a polypeptide encoded bya polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:59, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 59, or (iii) the full-length complement of (i) or(ii), wherein high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 59; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 60; wherein the saccharification of thecellulosic material is performed with the enzyme composition at atemperature in the range of about 40° C. to about 70° C.; (b) fermentingthe saccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.
 44. The method of claim43, wherein the enzyme composition further comprises a GH61 polypeptidehaving cellulolytic enhancing activity.
 45. The method of claim 44,wherein the GH61 polypeptide having cellulolytic enhancing activity isone or more polypeptides selected from the group consisting of: (a) apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 34 or 42; (b)a polypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 33 or 41, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 33 or 41, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 33 or 41; and (d) a polypeptide comprising the mature polypeptide ofSEQ ID NO: 34 or
 42. 46. The method of claim 43, wherein the enzymecomposition further comprises a Family 11 polypeptide having xylanaseactivity.
 47. The method of claim 46, wherein the xylanase is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 200; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 199 or its full-lengthcomplement, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamideand washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65°C.; (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 199; and (d) a polypeptidecomprising the mature polypeptide of SEQ ID NO:
 200. 48. The method ofclaim 43, wherein the enzyme composition further comprises one or morepolypeptides having endoglucanase activity.
 49. The method of claim 48,wherein the polypeptide has endoglucanase I activity and is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 20; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 19, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 19, or(iii) the full-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19; and (d) a polypeptide comprising the mature polypeptide of SEQID NO:
 20. 50. The method of claim 48, wherein the polypeptide hasendoglucanase II activity and is selected from the group consisting of:(a) a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 26; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 25, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 25, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 25; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO:
 26. 51. Themethod of claim 43, wherein saccharification of the cellulosic materialis performed with the enzyme composition at a temperature in the rangeof about 50° C. to about 65° C.
 52. A method of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition comprising: (I) a polypeptidehaving cellobiohydrolase I activity selected from the group consistingof: (a) a polypeptide comprising an amino acid sequence having at least95% sequence identity to the mature polypeptide of SEQ ID NO: 6; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 5, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 5, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 5; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO: 6; (II) apolypeptide having cellobiohydrolase II activity selected from the groupconsisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 18; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 17, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 17, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 18; (III) a polypeptide having beta-glucosidase activity selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 28; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 27, (ii) the cDNA sequence ofthe mature polypeptide coding sequence of SEQ ID NO: 27, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 27; and (d) a polypeptide comprising the mature polypeptide of SEQID NO: 28; and (IV) a Family 10 polypeptide having xylanase activity isselected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 95% sequence identity to the maturepolypeptide of SEQ ID NO: 48; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 47, (ii)the genomic DNA sequence of the mature polypeptide coding sequence ofSEQ ID NO: 47, or (iii) the full-length complement of (i) or (ii),wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide and washing threetimes each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 47; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 48; (V) a polypeptide having beta-xylosidaseactivity selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the mature polypeptide of SEQ ID NO: 60; (b) a polypeptide encoded bya polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:59, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 59, or (iii) the full-length complement of (i) or(ii), wherein high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (c) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 59; and (d) a polypeptide comprising the maturepolypeptide of SEQ ID NO: 60; wherein the saccharification of thecellulosic material is performed with the enzyme composition at atemperature in the range of about 40° C. to about 70° C.
 53. The methodof claim 52, wherein the enzyme composition further comprises a GH61polypeptide having cellulolytic enhancing activity.
 54. The method ofclaim 53, wherein the GH61 polypeptide having cellulolytic enhancingactivity is one or more polypeptides selected from the group consistingof: (a) a polypeptide comprising an amino acid sequence having at least95% sequence identity to the mature polypeptide of SEQ ID NO: 34 or 42;(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 33 or 41, (ii) the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 33 or 41, or (iii) thefull-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 33 or 41; and (d) a polypeptide comprising the mature polypeptide ofSEQ ID NO: 34 or
 42. 55. The method of claim 52, wherein the enzymecomposition further comprises a Family 11 polypeptide having xylanaseactivity.
 56. The method of claim 55, wherein the xylanase is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 200; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 199 or its full-lengthcomplement, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamideand washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65°C.; (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 199; and (d) a polypeptidecomprising the mature polypeptide of SEQ ID NO:
 200. 57. The method ofclaim 52, wherein the enzyme composition further comprises one or morepolypeptides having endoglucanase activity.
 58. The method of claim 57,wherein the polypeptide has endoglucanase I activity and is selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence having at least 95% sequence identity to the mature polypeptideof SEQ ID NO: 20; (b) a polypeptide encoded by a polynucleotide thathybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 19, (ii) the genomic DNAsequence of the mature polypeptide coding sequence of SEQ ID NO: 19, or(iii) the full-length complement of (i) or (ii), wherein high stringencyconditions are defined as prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide and washing three times each for 15 minutesusing 2×SSC, 0.2% SDS at 65° C.; (c) a polypeptide encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19; and (d) a polypeptide comprising the mature polypeptide of SEQID NO:
 20. 59. The method of claim 57, wherein the polypeptide hasendoglucanase II activity and is selected from the group consisting of:(a) a polypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 26; (b) apolypeptide encoded by a polynucleotide that hybridizes under at leasthigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 25, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 25, or (iii) the full-lengthcomplement of (i) or (ii), wherein high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%formamide and washing three times each for 15 minutes using 2×SSC, 0.2%SDS at 65° C.; (c) a polypeptide encoded by a polynucleotide comprisinga nucleotide sequence having at least 95% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 25; and (d) apolypeptide comprising the mature polypeptide of SEQ ID NO:
 26. 60. Themethod of claim 52, wherein saccharification of the cellulosic materialis performed with the enzyme composition at a temperature in the rangeof about 50° C. to about 65° C.